UBC Theses and Dissertations

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UBC Theses and Dissertations

Effects of feeding diethylstilbesterol and a forage antiestrogen on the reproduction of female mink.… Scheer, Howard Donald 1969

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I  II  The E f f e c t s o f F e e d i n g D i e t h y l s t i l b e s t e r o l and a Forage A n t i e s t r o g e n on the R e p r o d u c t i o n o f Female Mink. (Mustela v i s o n ) .  The E f f e c t s o f V a r i o u s P r o t e i n and Energy L e v e l s on the Maintenance and E a r l y Growth o f Mink. (Mustela v i s o n ) .  by  HOWARD DONALD SCHEER B.S.A., U n i v e r s i t y  o f B r i t i s h Columbia, 1966  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the D i v i s i o n of Animal S c i e n c e  required  We accept t h i s t h e s i s as conforming standard/  THE  UNIVERSITY OF BRITISH COLUMBIA APRIL 1969  t o the  In  presenting  an  advanced  the  degree  Library  I further for  this  shall  agree  scholarly  by  his  of  this  thesis  in p a r t i a l  f u l f i l m e n t of  at  University  of  the  make that  i t freely  permission  purposes  may  representatives.  written  thesis  for  It  financial  available for  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  flp ;I R  ^  J  Columbia  TFTF  by  the  is understood gain  for  extensive  granted  permission.  Department  Date  be  British  shall  reference  Head  be  requirements  Columbia,  copying  that  not  the  of  and  of my  I agree  for that  Study.  this  thesis  Department  copying  or  allowed  without  or  publication my  ABSTRACT  In P a r t A o f Study I , s i x groups of female standard  mink were f e d (once d a i l y ) e i t h e r 5, 10, o r  15 mcgm. of DES/mink/day o r a f o r a g e  antiestrogen  e x t r a c t a t l e v e l s o f 5, 10, o r 15 gm. e q u i v a l e n t o f alfalfa/mink/day.  I n P a r t B, seven groups o f female  p a s t e l mink were f e d (once d a i l y ) 15 mcgm. o f DES/mink/ day  p r i o r t o and a t v a r i o u s stages  period.  o f the g e s t a t i o n  A l s o , the a n t i e s t r o g e n e x t r a c t was again f e d  (once d a i l y ) a t l e v e l s o f 5, 10, o r 15 gm. e q u i v a l e n t of a l f a l f a t o t h r e e groups o f female p a s t e l mink a f t e r implantation of the f e r t i l i z e d From the r e s u l t s it  i s evident  egg had o c c u r r e d .  o f P a r t A and B o f Study I  t h a t DES a t l e v e l s as low as 5 t o 15 mcgm/  mink/day caused complete r e p r o d u c t i v e f a i l u r e .  A t the  15 mcgm. l e v e l i t i s p o s s i b l e t o prevent  or d i s r u p t  pregnancy i f the hormone i s a d m i n i s t e r e d  f o r at least  one week d u r i n g the p e r i o d s t a r t i n g one week b e f o r e breeding  and ending 30 days p r i o r t o p a r t u r i t i o n .  The  f o r a g e a n t i e s t r o g e n a t l e v e l s between 5 and 15 gm. equivalent litter  of a l f a l f a reduced the number o f k i t s p e r  from 5.8 t o 1.6.  The f e e d i n g o f v a r i o u s  levels  of DES o r the a n t i e s t r o g e n e x t r a c t d i d n o t adhere t o a dose/response r e l a t i o n s h i p e i t h e r i n i n t e r r u p t i n g pregnancy o r r e d u c i n g  the number of k i t s p e r l i t t e r .  The DES, f e d at l e v e l s of 5, 10, or 15 mcgm./mink/ day was a more p o t e n t a n t i f e r t i l l t y agent than the f o r a g e a n t i e s t r o g e n f e d at l e v e l s e q u i v a l e n t to 5, 10, or 15 gm. of a l f a l f a . In P a r t A o f S t u f y I I , 200 mature male and female mink of a v a r i e t y o f c o l o u r phases were d i v i d e d i n t o f i v e groups.  The groups were f e d (twice d a i l y )  r a t i o n s v a r y i n g i n crude p r o t e i n (36.6 t o 44.8%, on a d.m. basis).  b a s i s ) and gross f a t (21.3 t o 28.9%, on a I n P a r t B, 280 p a s t e l and s t a n d a r d  d.m.  kits  (6-7 wks. o l d ) were f e d (twice d a i l y ) r a t i o n s v a r y i n g i n crude p r o t e i n (39.0 to 54.3%, on a d.m. b a s i s ) and gross energy  (5.20 t o 5.52 Kcal/gm. dry f e e d ) .  From the r e s u l t s of P a r t A and B of Study I I it  i s evident  t h a t r a t i o n s c o n t a i n i n g 35 t o 40% crude  p r o t e i n and 20 t o 25% f a t , on a dry matter b a s i s , are s a t i s f a c t o r y f o r the l a t e growth requirements o f mink. c o e f f i c i e n t s obtained  and  maintenance  The mean apparent  digestibility  i n t h e s e s t u d i e s f o r dry m a t t e r ,  p r o t e i n , f a t , and energy a r e 66.38, 74.05, 87.20, and 73.74%, r e s p e c t i v e l y . energy  As the apparent  digestible  (A.D.E.) was r a i s e d from 353.4 Kcal/100 gm. dry  matter to 426.0 Kcal/100 gm. and as the apparent d i g e s t i b l e n i t r o g e n (A.D.N.) l e v e l reached 4.980 gm/100 gm  dry matter,  the e a r l y growth of b o t h male and female  mink was improved.  Maximum e a r l y growth r a t e was  o b t a i n e d i n k i t s when the r a t i o n c o n t a i n e d 85.54 K c a l of A.D.E./gm A.D.N. (13.6 K c a l gross energy/gm gross protein).  A l s o , i n c r e a s i n g A.D.E. content i n the  d i e t s had a d e f i n i t e p r o t e i n - s p a r i n g e f f e c t .  Maximum  n i t r o g e n r e t e n t i o n was o b t a i n e d when the d i e t  contained  426.0 K c a l o f A.D..E./100 gm. of dry f e e d . the r e s u l t s suggest  Finally  t h a t the growing male k i t s  (from 6-28  weeks of age) were capable of u t i l i z i n g feeds more e f f i c i e n t l y than female k i t s o f the same age.  ACKNOWLEDGEMENTS  The w r i t e r wishes t o express h i s s i n c e r e thanks t o Dr.  W.D.  K i t t s , Professor  o f Animal S c i e n c e  and Chairman  of the D i v i s i o n o f Animal S c i e n c e  for his  continued  encouragement and guidance d u r i n g  the course of t h i s  study.  A l s o , t h i s work was g r e a t l y b e n e f i t t e d by the a s s i s t a n c e and  c r i t i c i s m o f Mr. C l i f f o r d Harvey, Head T e c h n i c i a n of  the U.B.C. F u r Animal U n i t . due  Special appreciation i s also  t o Mrs. T a n n i s Warmer f o r h e r i n v a l u a b l e and u n t i r i n g  a s s i s t a n c e i n the l a b o r a t o r y . The  help  o f t h e many students who a s s i s t e d a t v a r i o u s  stages of t h e study i s a l s o g r a t e f u l l y  acknowledged.  TABLE OF CONTENTS PAGE  The E f f e c t s o f Feeding D i e t h y l s t i l b e s t e r o l and a Forage A n t i e s t r o g e n on t h e Reproduction of Mink, (Mustela v i s o n ) . I.  Introduction  1  II.  L i t e r a t u r e Review A. Estrogens and A n t i e s t r o g e n s  2 2  1)  Suppression  2)  The e f f e c t s o f estrogens and a n t i e s t r o g e n s on t u b a l t r a n s p o r t . a) estrogens b) antiestrogens c) mechanism o f a c t i o n i) estrogens ii) antiestrogens  4 4 8 10 10 13  The e f f e c t s o f estrogens on i m p l a n t a t i o n .  16  3) 4) 5)  III.  of p i t u i t a r y gonadotrophins.  and a n t i e s t r o g e n s  The e f f e c t s o f estrogens and a n t i e s t r o g e n s on pregnancy a f t e r i m p l a n t a t i o n has o c c u r r e d .  20  The d i r e c t e f f e c t s of estrogens antiestrogens.  22  and  M a t e r i a l s and Methods. A. Feeding T r i a l - P a r t A. 1)  Materials a) b)  2)  Feeding  23 23 23  animals housing  Methods a) b) c) d) e)  B.  3  23  feeding e x t r a c t i o n of antiestrogens antiestrogen a c t i v i t y o r a l a c t i v i t y o f DES. s t a t i s t i c a l analysis T r i a l - P a r t B.  24  1)  Materials a) animals b) housing  25  2)  Methods a) feeding b) e x t r a c t i o n of antiestrogens c) antiestrogen a c t i v i t y d) s t a t i s t i c a l analysis  25  IV.  Results  and D i s c u s s i o n  V.  Conclusions.  VI.  Bibliography  V I I . Appendices  The E f f e c t s of V a r i o u s P r o t e i n and Energy L e v e l s on the E a r l y Growth and Maintenance of Mink, (Mustela vison). I.  Introduction  II.  L i t e r a t u r e Review A. R e l a t i o n s h i p of P r o t e i n u t i l i z a t i o n Energy Intake.  B.  1)  The e f f e c t s of constant v a r y i n g energy i n t a k e .  p r o t e i n with  2)  The e f f e c t s of constant energy w i t h varying protein intake.  The e f f e c t s of Carbohydrate and F a t on Protein Utilization.  I I I . M a t e r i a l s and Methods. A. Feeding T r i a l - P a r t A.  B.  to  1)  Materials a) animals b) housing c) feed  2)  Methods a) animals b) feeds c) s t a t i s t i c a l analysis  Study I I - P a r t B. 1)  Feeding T r i a l a) Materials i) animals ii) housing iii) feed b)  Methods  PAGE  2)  Digestibility Trial a) Materials i) animals ii) digestibility iii) feed  52 52 cages  Methods  53  and D i s c u s s i o n  54  b) IV.  Results  V.  Conclusions  64  VI.  References c i t e d  65  V I I . Appendices  77  LIST OF TABLES  TABLE  PAGE  TABLE I  A n t i e s t r o g e n i c a c t i v i t y o f the a l f a l f a e x t r a c t used i n Study I P a r t A  27  TABLE I I  A n t i e s t r o g e n i c a c t i v i t y o f the a l f a l f a e x t r a c t used i n Study I P a r t B  28  TABLE I I I  The e s t r o g e n i c a c t i v i t y o f DES a d m i n i s t e r e d o r a l l y t o w e a n l i n g female wistar rats  29  TABLE IV  R e p r o d u c t i v e performance o f mink r e c e i v i n g DES o r a n t i e s t r o g e n a f t e r ova i m p l a n t a t i o n - P a r t A  32  TABLE V  R e p r o d u c t i v e performance of mink r e c e i v i n g DES a t v a r i o u s stages o f pregnancy o r a n t i e s t r o g e n a f t e r implantation  35  TABLE VI  G e n e r a l composition  46  TABLE V I I  The composition of P a r t A  TABLE V I I I  The composition o f e x p e r i m e n t a l mixes o f P a r t A  cereal  48  TABLE IX  The composition of e x p e r i m e n t a l mixes o f P a r t B  cereal  51  TABLE X  Mean i n i t i a l , f i n a l and body weight gains o f Study I I , P a r t A  TABLE XI  Proximate a n a l y s i s of r a t i o n s f o r Study I I , P a r t A  TABLE X I I  Proximate a n a l y s i s o f r a t i o n s f o r Study I I , P a r t B  of r a t i o n s  of the c o n t r o l c e r e a l  47  54  v.  55  56  LIST OF TABLES (continued)  PAGE TABLE X I I I  Mean i n i t i a l , f i n a l and body weight gains o f Study I I , P a r t B  TABLE XIV  Summary o f d i g e s t i b i l i t y  trial  TABLE XV  Summary o f d i g e s t i b i l i t y  trial II .  TABLE XVI  The apparent d i g e s t i b l e energy: apparent d i g e s t i b l e n i t r o g e n r a t i o f o r the e x p e r i m e n t a l r a t i o n s as determined from d i g e s t i b i l i t y t r i a l II  TABLE XVII  The p r o t e i n - s p a r i n g e f f e c t of i n c r e a s i n g apparent d i g e s t i b l e energy  I ..  57  59  60  61  LIST OF FIGURES PAGE  FIG.I  The dose/response r e l a t i o n s h i p between o r a l a d m i n i s t r a t i o n o f DES and u t e r i n e weight of an immature o v a r i e c t o m i z e d Wistar r a t .  30  FIG.II  R e l a t i o n s h i p between p e r c e n t A.D.N, and the A.D.E. :A..D.N. r a t i o .  62  LIST OF APPENDICES  APPENDIX  PAGE  APPENDIX I  Antiestrogen  extraction  APPENDIX I I  Bioassay o f E s t r o g e n i c and A n t i e s t r o g e n i c compounds  78  APPENDIX I I I  I n i t i a l and F i n a l Body Weights o f Study I I , P a r t A  80  APPENDIX IV  I n i t i a l and F i n a l Body Weights o f Study I I , P a r t B  83  APPENDIX V  Digestibility  Trial I  90  APPENDIX VI  Digestibility  Trial II  95  APPENDIX V I I  A n a l y s i s of Variance Covariance Tables  and  77  100  STUDY I  The and  E f f e c t s of Feeding  Diethystilbesterol  a Forage A n t i e s t r o g e n on t h e R e p r o d u c t i o n  (Mustela  vison).  PART A.  After  I m p l a n t a t i o n of Ova.  PART B.  B e f o r e I m p l a n t a t i o n of Ova.  o f Mink,  I.  Introduction  I t has become e v i d e n t t h a t v a r i o u s hormones o f animal and p l a n t o r i g i n and c e r t a i n s y n t h e t i c hormones have a marked e f f e c t on f e r t i l i t y .  Occasionally,i n  the p a s t , the c o n t a m i n a t i o n of feeds by estrogens has caused r e p r o d u c t i v e d i s t u r b a n c e s r e s u l t i n g i n a decrease in  the number o f l i t t e r s  i n b r e e d i n g c o l o n i e s of  142 laboratory animals.  I n commercial e n t e r p r i s e s , the  a r e a which has been most d r a s t i c a l l y a f f e c t e d by e s t r o g e n c o n t a m i n a t i o n o f feeds i s the mink i n d u s t r y . s y n t h e t i c estrogen, d i e t h y l s t i l b e s t e r o l  The  (DES), f o r a  number o f y e a r s , caused marked r e d u c t i o n s i n the annual kit  p r o d u c t i o n a c r o s s Canada and the U n i t e d S t a t e s .  During  the l a t e 1950's, the i n d i s c r i m i n a t e use of DES as a "growth s t i m u l a n t " r e s u l t e d i n the i n t r o d u c t i o n of DES i n t o mink feeds v i a p e l l e t - i n s e r t e d  chicken h e a d s , p o u l t r y  viscera  105 or  d r i e d tankage,  and beef t r i p e from animals 136  implanted  w i t h or f e d DES p r i o r t o s l a u g h t e r . Today, however, l e g i s l a t i o n a t the t u r n of the decade has  l i m i t e d the use of DES i n l i v e s t o c k f e e d i n g .  r e s u l t , l e s s c o n t a m i n a t i o n of s l a u g h t e r h o u s e  As a  by-products,  e s p e c i a l l y i n the p o u l t r y i n d u s t r y , has safeguarded a g a i n s t the major i n t r o d u c t o r y r o u t e s of DES i n t o mink r a t i o n s . The d r a s t i c e f f e c t s of estrogens on f e r t i l i t y ,  however,  causes concern t h a t o t h e r p o s s i b l e r o u t e s of e s t r o g e n contamination  exist.  still  L i t e r a t u r e Review A.  E s t r o g e n s and  Antiestrogens  R e p r o d u c t i v e d i f f i c u l t i e s i n animals  grazing  c e r t a i n k i n d s of p a s t u r e s were f i r s t n o t i c e d A u s t r a l i a i n c o n n e c t i o n w i t h the i n c r e a s e d subterranean clover sheep pasture."'""'" like  use  plants.  ( T r i f o l i u m subterranean) as S i n c e then, a number of  a  estrogen-  96. '  '  The  most s i g n i f i c a n t p l a n t  estrogen  c o u m e s t r o l i s o l a t e d from l a d i n o c l o v e r ( T r i f o l i u m  rep ens)  by B i c k o f f , et a l " ^ i n 1957.  K i t t s , et a l ^ '  demonstrated t h a t the l e v e l of e s t r o g e n i c c e r t a i n legumes and c l o v e r , and  and  of  compounds have been i s o l a t e d from a v a r i e t y o f 95  is  in  grasses  (e.g.  b i r d s f o o t t r e f o i l ) was  decreased toward autumn.  t h a t the  alfalfa, highest  a c t i v i t y may  w i t h the r e p r o d u c t i v e  status  in  ladino i n spring 80  Legg, et_ a l  l e v e l of e s t r o g e n i c  i n some way  activity  ^  felt  be  associated  of the  plant  m a t e r i a l from which they were r e c o v e r e d . I n 1960,  B i c k o f f , et_ a l ^  found i n h i b i t o r s to  e s t r o g e n a c t i v i t y i n some a l f a l f a samples. i n 1962  Adler,"'"  demonstrated t h a t as w e l l as p o s s e s s i n g  estrogenic  p r o p e r t i e s , a l f a l f a a l s o contained a n t i e s t r o g e n i c 97 properties.  O s t r o v s k y and  birdsfoot t r e f o i l  contains  Kitts  have suggested  an a n t i e s t r o g e n  that  s i m i l a r to  31  t h a t found i n a l f a l f a . r e p o r t e d the presence  Cook and K i t t s  33  have  of an a n t i e s t r o g e n i c substance 2  i n yellow pine needles. the presence hay  I n 1965,  established  of e s t r o g e n i n h i b i t o r y a c t i v i t y i n oat  (Avena s a t i n a ) and f a h l i  alexandrinum  Adler  var.  c l o v e r hay  (Trifolium  Fahli).  I n h i b i t i o n of f e r t i l i t y by estrogens and estrogens  anti-  can be a c h i e v e d by i n t e r f e r i n g w i t h normal  mechanisms at any one  of s e v e r a l v u l n e r a b l e p o i n t s i n 114 the r e p r o d u c t i v e p r o c e s s . These i n c l u d e impairment .  of  gametogenesis by 72  compounds h a v i n g d i r e c t a c t i o n  on  112  germ c e l l s ,  '  p r e v e n t i o n of o v u l a t i o n by a d m i n i s t e r i n g  s t e r o i d s which suppress p i t u i t a r y p r o d u c t i o n of gonadotrophins progesterone  '"^ i n t e r f e r e n c e w i t h the e s t r o g e n /  r a t i o s e s s e n t i a l f o r t u b a l t r a n s p o r t and  i m p l a n t a t i o n of the f e r t i l i z e d  ovum and d e s t r u c t i o n of  ova and d e v e l o p i n g embryos w i t h a n t i m e t a b o l i t e s . 1)  S u p p r e s s i o n of P i t u i t a r y The  antifertility  Gonadotrophins  e f f e c t s due  to prevention  of  o v u l a t i o n can be o b t a i n e d by c h e m i c a l antagonism 72 to the s e c r e t i o n of gonadotrophins. Compounds which can antagonize the s e c r e t i o n of gonadotrophins i n c l u d e e s t r o g e n s , such as DES,  72  and  norethynodrel;  androgens, such as e t h i n y l a n d r o s t e n e d i o l lproprionate  13  112  3-cyclohexy-  ; and.the p r o g e s t a t i o n a l s t e r o i d s .  72  The i n h i b i t i o n of the p i t u i t a r y  gonadotrophin  s e c r e t i o n ^ r e s u l t s i n an impairment development and subsequent  of  follicular  o v u l a t i o n of the  103 mature ovum. The e f f e c t s of estrogens and a n t i e s t r o g e n s on t r a n s p o r t of the f e r t i l i z e d ovum. a)  Estrogens F o l l o w i n g the i n i t i a l work of Parkes and B e l l e r b y ,  98  and Smith  130  w i t h mice and  r a t s r e s p e c t i v e l y and K e l l y ^ w i t h  guinea  21 p i g s , B u r d i c k and Pincus  i n 1934  suggested  t h a t the d a i l y i n j e c t i o n of an e s t r o g e n ( e o t r i n ) i n t o mice and r a b b i t s d u r i n g the p r e i m p l a n t a t i o n p e r i o d r e s u l t e d i n the r e t e n t i o n of ova i n the F a l l o p i a n I t was was  tubes.  f e l t t h a t the r e t e n t i o n of the  p r o b a b l y due  ova  t o e i t h e r a c l o s u r e of the  t u b o - u t e r i n e j u n c t i o n or an a l t e r a t i o n i n the n a t u r e of the c i l i a r y movement which s u b s e q u e n t l y r e s u l t e d i n the i n h i b i t i o n of 21 pregnancy.  A l s o , B u r d i c k and P i n c u s  found  t h a t a l l ovay whether they descended i n t o  the  u t e r u s o r remain i n the t u b e s , showed d e f i n i t e s i g n s o f d e g e n e r a t i o n by the f o u r t h day  copulation.  I n 1936,  Whitney and  after 138  Burdick  showed t h a t the i n j e c t i o n of e s t r o g e n i c substances  d u r i n g the p r e i m p l a n t a t i o n p e r i o d  5-.  caused r e t e n t i o n of ova tubes and  i n the F a l l o p i a n  o r i g i n a t e d the term  "tube-locking" 138  of ova.  Whitney and  tube-locking  occurred  Burdick  found t h a t  when low  doses (5-10  of an e s t r o g e n i c substance was rat,  but  injected into  suggested t h a t w i t h h e a v i e r  estrogens increased to increased  ova  transport  ciliary motility.  t o 500  R.U.  occurred  due  I t was l a t e r 23 24 '  of an  that  estrogenic  substance a c c e l e r a t e d the passage of ova  fertilized  through the F a l l o p i a n tubes of mice.  were f r e q u e n t l y found i n the uterus two-celled  stage,  vaginal plug.  than u s u a l . obtained  30  Ova  even i n the  t o 40 h r s . a f t e r f i n d i n g the  T h i s i s a t l e a s t 30 hours sooner 139 I n 1938,  Whitney and  Burdick  s i m i l a r results after i n j e c t i n g rabbits  w i t h a s i n g l e massive dose (5000 R.U.) e s t r o g e n i c substance. as DES,  the  doses of  demonstrated by B u r d i c k and Whitney i n j e c t i o n s of 100  R.U.^)  of  an  Synthetic estrogens,  such  administered  i n e a r l y pregnancy a l s o 22 a c c e l e r a t e d the r a t e of t u b a l passage. To i n v e s t i g a t e f u r t h e r the e f f e c t s of estrogens 44 on pregnancy, D r e i s b a c h ,  i n 1959,  attempted  f i n d the s m a l l e s t dose of hormone and 1. R.U. = r a t u n i t .  the most  to  6.  appropriate  time of a d m i n i s t r a t i o n which would  produce a l t e r a t i o n s i n t u b a l t r a n s p o r t i n the rat.  He found t h a t as l i t t l e  as 0.02 mg/kg.  body weight was e f f e c t i v e d u r i n g  the p e r i o d  of t u b a l t r a n s p o r t and the p e r i o d most s e n s i t i v e to e s t r o g e n a d m i n i s t r a t i o n was the t h i r d day 58 post-coitum.  A l s o i n 1959, Greenwald  studied  the comparative e f f e c t i v e n e s s o f estrogens i n i n t e r r u p t i n g pregnancy i n the r a b b i t .  He found  t h a t e s t r a d i o l benzoate was the most e f f e c t i v e e s t r o g e n i n i n t e r r u p t i n g pregnancy when compared with estrone  and DES.  In 1961"^ and a g a i n i n 1 9 6 3 ^ Greenwald, i n h i s study of the t r a n s p o r t rabbit oviduct,  reported  of ova through t h e  r e s u l t s which were  contrary  to t h e t u b e - l o c k i n g  e f f e c t o f e s t r o g e n s i n the r a t 21 r e p o r t e d by Whitney and B u r d i c k i n 1936 and the a c c e l e r a t i o n e f f e c t on f e r t i l i z e d ova r e p o r t e d by 59 B u r d i c k and Whitney i n 1937.  Greenwald  reported  t h a t w h i l e the i n j e c t i o n o f 250 pg o f e s t r a d i o l r e s u l t e d i n t h e tube r e t e n t i o n of ova, 25 ;yg o f the same compound a c c e l e r a t e d ova t r a n s p o r t i n t o the uterus;  vagina.  many o f the ova were then e x p e l l e d i n t o the 59 A l s o , Greenwald  found t h a t when t h e ova  were r e t a i n e d i n the tube by the i n j e c t i o n of a l a r g e  7:  dose of e s t r o g e n , the ova were b l o c k e d , n o t a t the t u b o - u t e r i n e j u n c t i o n as r e p o r t e d  earlier,  21  but a t the b e g i n n i n g of the isthmus or a m p u l l a r y 60 isthmus j u n c t i o n . found  L a t e r i n 1961, Greenwald  t h a t a f t e r a s i n g l e i n j e c t i o n of e s t r a d i o l  c y c l o p e n t y l p r o p i o n a t e , on the morning sperm were found  i n the v a g i n a , t u b a l and u t e r i n e m o t i l i t y  were i n c r e a s e d so t h a t the f e r t i l i z e d e x p e l l e d from the u t e r u s by 48 h r s .  eggs were  post-coitum.  40 Deanesly,  i n 1963, c o n c u r r e n t l y found  t h a t low  doses of e s t r a d i o l benzoate, g i v e n immediately mating caused  premature e x p u l s i o n of f e r t i l i z e d  from the F a l l o p i a n tubes suggested dropped.  after  i n the guinea p i g .  eggs  He  t h a t the term " t u b e - l o c k i n g " s h o u l d be I n 1964, Banik  and P i n c u s ^ showed t h a t  as much as 20 u g / r a t of e s t r o n e a d m i n i s t e r e d on Day 1 of  pregnancy e x p e l l s the eggs from the tube i n t o the  u t e r u s i n about 20-24 hours on Day 2. evidence  t h a t a l a r g e q u a n t i t y of e s t r o n e  b l o c k s the f e r t i l i z e d  A l s o i n 1964, Harper, to  There was no (250 ug/rat)  egg at the u t e r o - t u b a l j u n c t i o n . 64 using autoradiographic  t r a c e the movement of " a r t i f i c i a l  eggs"  techniques  (radioactive  s p h e r e s ) , demonstrated t h a t e s t r a d i o l benzoate, i n the dose a d m i n i s t e r e d  (0.5 y g ) , had an a c c e l e r a t i n g e f f e c t on  the passage o f the spheres  through  the tube, and a l s o  8.  through the u t e r u s . despite  I t was observed, however, t h a t  the a c c e l e r a t i n g e f f e c t o f the e s t r o g e n a  c e r t a i n p e r c e n t a g e o f spheres were t r a p p e d a t the j u n c t i o n of the ampulla and the isthmus. Humphrey and M a r t i n  ^  i n 1968,  p r e s e n t e d experiments i n which one  l e v e l of e s t r a d i o l - 1 7 3 and  Recently,  (0.4 yg/day) caused both r e t e n t i o n  l o s s of ova i n mice.  Retention  d i d not only  involve  blockage a t t h e u t e r o - t u b a l j u n c t i o n , s i n c e ova were a l s o l o c a t e d i n the ampulla.  L a t e r i n 1968, Humphrey^  demonstrated t h a t s p h i n c t e r s  a t the ampulla-isthmus and  isthmo-uterine  junctions  (utero-tubal)  delaying f e r t i l i z e d Fallopian  ova d u r i n g  a r e capable of  t h e i r sojourn  through the  tubes.  A n t i e s trogens Although n o t as e x t e n s i v e l y s t u d i e d as the b o t h the n a t u r a l and s y n t h e t i c a n t i e s t r o g e n s  estrogens,  a r e capable  o f i n t e r f e r i n g w i t h pregnancy by a l t e r i n g the r a t e of t u b a l transport  of the f e r t i l i z e d  ovum.  I t was f i r s t  reported  20 by B u r d i c k ,  Emmerson  testosterone mice w i l l The  and Whitney,  i n 1940 t h a t  p r o p i o n a t e i n j e c t e d i n adequate amounts i n t o  cause the r e t e n t i o n of b l a s t o c y s t s i n the o v i d u c t s .  r e s u l t s showed t h a t 2.0 mg. d a i l y was the l i m i n a l  amount of t e s t o s t e r o n e  p r o p i o n a t e n e c e s s a r y t o cause  r e t e n t i o n of b l a s t o c y s t s i n the tubes beyond 100 h r s . a f t e r 59 the  formation  o f the v a g i n a l p l u g .  I n 1961, Greenwald  w i t h a s i n g l e i n j e c t i o n of p r o g e s t e r o n e caused some a c c e l e r a t i o n i n ova t r a n s p o r t .  In o n l y one i n s t a n c e were  9-  ova recovered from the uterus at 48 hrs. a f t e r administration of 25 mg. of progesterone.  Acceleration  of ova was also obtained with the i n j e c t i o n of similar amounts of progestin. 64 In 1964, Harper,  found that progesterone delayed  movement of the radioactive spheres through the ampulla very markedly, but i f the spheres reached the isthmus they 30 passed into the uterus on a l l occasions. Chang i n 1964 31 and Chang and Yanagimachi  i n 1965 showed that a number of  antiestrogens interfered with ovum transport i n rabbits, causing acceleration i n some and retention i n others.  In  1968, Humphrey and M a r t i n ^ demonstrated that DMS * (100yg/day), 1  1  MER-25 ' (2.0 mg/day) and MRL-37 '(2.0 mg/day) administered 2  3  subcutaneously on Days 1, 2 and 3 post-coitum to female mice resulted i n retention of ova i n the oviduct.  As w e l l ,  the antiestrogens delayed the development of the retained ova and ova were prominently recovered from the tube i n the morula rather than the b i a s t u l a stage. Although antiestrogens are capable of disrupting the passage of f e r t i l i z e d ova through the Fallopian tubes, they are even more e f f e c t i v e as anti-implantation agents.  In  44 1959, Dreisbach  1. 2. 3.  reported that pregnant rats were most  DMS = dimethylstilbesterol MER-25 = l-(p-2-diethylamino-ethoxyphenyl)-lphenyl-2-anisyl ethanol MRL-37 = corresponding ethane of MER-25  i d . -  s e n s i t i v e t o a n t i e s t r o g e n a d m i n i s t r a t i o n thus from the-itime when e s t r o g e n s produce  differs  their greatest effect,  t h a t i s when t h e ova a r e i n the F a l l o p i a n  tubes.  Mechanism o f A c t i o n : i)  Estrogens With regards t o t u b e - . l o c k i n g , i t i s p r e s e n t l y  felt  t h a t the a m p u l l a r y - i s t h m i c j u n c t i o n r a t h e r than the u t e r o t u b a l j u n c t i o n i s the primary b l o c k t o egg t r a n s p o r t i n the rabbit.^  A c c o r d i n g t o Wintenberger-Torr^s''"^''" the major  b l o c k t o egg t r a n s p o r t i n the ewe occurs a t t h i s  junction.  61  But Greenwald  f e e l s t h a t the u t e r o - t u b a l j u n c t i o n i s  p r o b a b l y the primary b a r r i e r i n some s p e c i e s . I t i s important t o comprehend the n a t u r e of the b l o c k i n g mechanisms n o t only t o understand how l a r g e doses of 61  e s t r o g e n i c compounds a c t t o r e t a i n ova i n the tube, b u t a l s o t o determine how low l e v e l s of e s t r o g e n s prematurely advance the time of opening of the a m p u l l a r y - i s t h m i c 61  junction.  The mechanism of a c t i o n may a l s o e x p l a i n why 138  Whitney and B u r d i c k  found t h a t low doses  r e s u l t e d i n t u b e - l o c k i n g and l a r g e doses  o f an e s t r o g e n  caused ova  a c c e l e r a t i o n ; r e s u l t s which a r e c o n t r a d i c t o r y t o the more recent  reports.  The  first  t h e o r y proposed was by H a r t m a n ^ i n 1 9 3 9 .  Hartman suggested t h a t t h e a d m i n i s t r a t i o n o f an e s t r o g e n caused an a l t e r a t i o n i n the e s t r o g e n - p r o g e s t e r o n e b a l a n c e ,  11.  which caused a v a r i a t i o n i n the r a t e and amplitude of muscle a c t i v i t y i n the tube and i n t u r n the r a t e o f passage of the ova. A second t h e o r y was proposed by A l d e n ^ i n 1942. From h i s o b s e r v a t i o n s that while c i l i a r y  by t r a n s i l l u m i n a t i o n , A l d e n  felt  a c t i o n was the p r i m a r y f o r c e i n  e f f e c t i n g the e n t r a n c e of the egg i n t o and through the cephalic ampulla, a c t i o n o f the t u b a l musculature was t h e c o n t r o l l i n g f a c t o r governing f u r t h e r advancement.  The  administration  of an e s t r o g e n , t h e r e f o r e , would r e s u l t i n the c l o s u r e o f the  lumen s p h i n c t e r  a t the s i t e o f t h e b l o c k  due t o the  c o n t r a c t i o n of muscle l a y e r s . 16 B l a c k and A s d e l l  17 '  l a t e r proposed a t h i r d  t o account f o r the r e t e n t i o n of the f e r t i l i z e d  theory  ova.  They  suggested t h a t t h e r e was a s l i g h t o r complete absence of a c t i v i t y o f the c i r c u l a r muscle a t t h e s i t e of the b l o c k e d ova.  A l s o movement i n d u c e d by the c i r c u l a r muscle i s  sufficient the  oviduct  t o account f o r t r a n s p o r t  o f ova through a l l o f  except the isthmus p o r t i o n immediately  t o the t u b o - u t e r i n e  junction.  anterior  The absence o f c i r c u l a r  muscle a c t i v i t y a t t h e u t e r i n e end o f the o v i d u c t  may  p a r t i a l l y account f o r ovum delay i n t h i s p o r t i o n of the 17 17 tube. B l a c k and A s d e l l have attempted t o demonstrate the p r e s e n c e of edema i n the t u b o - u t e r i n e  j u n c t i o n , but  12.  these attempts have f a i l e d .  They f e e l , however, that  the p o s s i b i l i t y s t i l l exists that i t i s present  since  the degree of edema would not have to be great to be e f f e c t i v e i n c l o s i n g the tube. 46 Edgar and A s d e l l ,  i n 1960, from t h e i r study of the  v a l v e - l i k e action of the utero-tubal junction i n the ewe, were able to propose a fourth theory.  Evidence they  presented indicates that estrogens cause edema on the subserosa and muscle coats of the w a l l of the utero-tubal junction of the ewe.  I t seems l i k e l y that the edema  causes an increase i n the degree of flexure of the tubal wall i n the immediate area and the two phenomena  contribute,  along with the narrow lumen and the folding of the tubal 46 l i n i n g to the v a l v e - l i k e action of the utero-tubal junction. 93 In support of this theory, Voyes, et a l  found i t  increasingly d i f f i c u l t to force f l u i d from the ampulla through the utero-tubal junction as the dosage of estrogen was increased. F i n a l l y a f i f t h theory was proposed by Wintenburger141 Torres  i n 1961 which suggests that strong  antiperistaltic  contractions of the isthmus maintain the egg i n the ampulla and prevent any further progression down the uterine tube. At present these theories.  there i s l i t t l e evidence to support any of I t would appear, however, that the i n j e c t i o n  of estrogen i n t e r f e r e s with progesterone dominated 23 57 mechanisms. Greenwald i n 1957 demonstrated that mucin, a mucopolysaccharide, secreted by the tubal epithelim onto the ova i s i n h i b i t e d by the administration of e s t r a d i o l i n rabbits.  I t i s not known, however, whether or not the  ¥3*.  reduced mucin l a y e r p l a y s r e t e n t i o n of f e r t i l i z e d In c o n c l u s i o n  a significant role in ova.  ovum t r a n s p o r t i s a i d e d by  c o n t r a c t i l e a c t i v i t y of the tube which i s by  a n a t o m i c a l and  the  co-ordinated  p h y s i o l o g i c a l mechanisms.  the mechanisms of r e c e p t i o n and markedly a f f e c t e d by  the  transport  the complex a c t i o n s  between the o v a r i a n s t e r o i d s and  Many of  of ova and  are  interactions  p i t u i t a r y hormones, the 63  sympathetic nervous system and Any  the a d r e n a l  steroids.  abnormal a l t e r a t i o n s i n hormone r a t i o s ,  therefore,  would cause a l t e r a t i o n s i n both r e c e p t i o n and of the ii)  fertilized  transport  ova.  Antiestrogens To  determine the mechanismsby which  i n t e r r u p t pregnancy, i t was  first  antiestrogens  n e c e s s a r y t o determine  whether these compounds were a c t i n g e i t h e r by v i r t u e of their estrogenic  or a n t i e s t r o g e n i c p r o p e r t i e s . 87  shown by M a r t i n ,  et a l  estrogenic  i n 1960  (or p r o - e s t r o g e n i c )  and h i s c o l l e a g u e s  It  that antiestrogens  i n high  doses.  was  are  also  Martin  d i d , however, show t h a t d i f f e r e n c e s e x i s t  between the r e l a t i v e potency of d i m e t h y l s t i l b e s t e r o l , the most p o t e n t a n t i - s t r o g e n fertility  agents and  t e s t e d and  e s t r a d i o l as  t h e i r r e l a t i v e potency as  antiestrogens.  A l s o , d i f f e r e n c e s e x i s t e d between t h e i r c a p a c i t i e s t o i n t e r r u p t pregnancy i n s i n g l e doses. concluded t h a t DMS,  I t was  therefore  a t l e a s t , p r o b a b l y a c t s by v i r t u e of i t s  14.  antiestrogenic properties.  In the same year, Emmens,et a l  found that when injected together, DMS t h e a n t i - f e r t i l i t y action of e s t r a d i o l . as very good evidence that DMS proestrogen i n preventing  did not antagonize This was  regarded  i s , i n f a c t , acting as a  fertility.  demonstrated that a number of synthetic interrupted early pregnancy i n mice.  In 1963,  Martin, et a l  anti-estrogens I t was  s t i l l not c l e a r ,  however, whether they did so by reason of t h e i r a n t i estrogenic or proestrogenic  a c t i v i t y since, i n the series  tested, both properties were highly correlated with the effectiveness of the compound i n i n t e r r u p t i n g pregnancy. 52 Again i n 1964,  Emmens, et a l  while studying  the  estrogenic  and antiestrogenic a c t i v i t y of compounds related to found a high c o r r e l a t i o n between proestrogenic, estrogenic and a n t i - f e r t i l i t y potencies I t was  DES,  anti-  i n these compounds.  s t i l l hazardous, however, from the evidence presented,  to ascribe the a n t i - f e r t i l i t y action of antiestrogens either t h e i r proestrogenic  to  or antiestrogenic properties.  Up u n t i l t h i s point the majority of the evidence presented was  interpreted i n favour of DMS  acting as an  a n t i - f e r t i l i t y agent by v i r t u e of i t s antiestrogenic 133 activity.  However, i n 1964,  Stone and Emmers,  found that  s e n s i t i v i t y to single injections of both e s t r a d i o l and was  highest approximately 72 hrs. a f t e r mating and  decreased after 96 hrs.  DMS  had  In view of the s i m i l a r action of the  two compounds and the lack of consistent antagonism when  15.  injected together, i t was suggested that the a n t i f e r t i l i t y action of DMS i n the mouse was due to i t s pro-estrogenic a c t i v i t y .  In a second f e r t i l i t y  study,  134 Stone and Emmens  found that a s i n g l e , simultaneous  i n j e c t i o n of DMS and e s t r a d i o l to 60 hrs. pseudopregnant rats showed only an additive effect i n i n h i b i t i n g deciduoma formation and suggested that DMS was acting as a proestrogen 49 50 rather than an antiestrogen.  In 1965,  Emmens,  '  by  further detailed analysis of the a c t i v i t i e s of DMS by d i f f e r e n routes of administration i n the rat and mouse, decided that i n the case of that p a r t i c u l a r compound the a n t i - f e r t i l i t y effect i s very probably due to i t s proestrogenic action. However, among the non-steroids, some, including MRL-37 and MER-25 may have a n t i - f e r t i l i t y effects unrelated or incompletely related to estrogenic a c t i v i t y and possibly related to antiestrogenic a c t i v i t y . F i n a l l y , i n 1968, Humphrey and M a r t i n ^ presented results which confirm the view that most antiestrogens are acting by v i r t u e of t h e i r proestrogenic a c t i v i t y .  16.  The  e f f e c t s of estrogens  The  ability  and  antiestrogens  of estrogens  pregnancy, a d m i n i s t e r e d  and  on  implantation.  anti-estrogens  a f t e r the ovum has  to i n t e r r u p t  normally  u t e r u s , has been w e l l d e s c r i b e d i n the l i t e r a t u r e .  entered  the  I n 1938, .  99 Parkes, et a l  demonstrated t h a t o r a l a d m i n i s t r a t i o n of  i s e f f e c t i v e i n preventing prepared  estrogens  i m p l a n t a t i o n of the b l a s t o c y s t .  by Dodds, Goldberg, Lawson and Robinson  42  , the  DES,  biological  p r o p e r t i e s of which have been d e s c r i b e d by Dodds, Lawson and is  r e p o r t e d h i g h l y a c t i v e by mouth.  highly e f f e c t i v e i n preventing  Noble,  O r a l a d m i n i s t r a t i o n of DES  i m p l a n t a t i o n i n r a t s at l e v e l s as  43  is low  99 as 5 t o  16ug. 36  D'Amour, et a l ,  i n 1933,  presented  evidence  a p p r o p r i a t e dosages of e s t r i n , a d m i n i s t e r e d of g e s t a t i o n , prevented was  not  i n d i c a t i n g that  d u r i n g the e a r l y  i m p l a n t a t i o n i n the r a t and  that t h i s  overcome by simultaneous i n j e c t i o n of an e s t r o g e n 37  such as progesterone.  In 1934,  D'Amour and  t h a t the a c t i o n of e s t r i n might be  mucosa i n such a manner a s t t o prevent Greenwald,"*^ i n 1957, estrogens  has  Gustavson  a d i r e c t one,  antagonist,  suggested  a f f e c t i n g the u t e r i n e  r e p o r t e d t h a t the a d m i n i s t r a t i o n of i n v o l v e s an  alteration  i n the p r o g e s t e r o n e dominated u t e r i n e environment prevents 114 S e g a l and Nelson  MER-25 can a l s o i n t e r f e r e w i t h  effect  implantation.  i n the r a b b i t 72 h r s . p o s t - c o i t u m  i m p l a n t a t i o n of ova.  stages  reported  the  t h a t the  the p r o g e s t a t i o n a l support  of  anti-estrogen the  endometrium, which r e s u l t s i n the reduced development of i m p l a n t a t i o n  sites,  ;i7.  Drelsback  31 44 ' found t h a t as l i t t l e  as 0.1 mg/kg. body weight  of e s t r o n e , i n j e c t e d s u b c u t a n e o u s l y , was n e c e s s a r y t o prevent i m p l a n t a t i o n i n the r a t when the ova were i n the u t e r u s .  Fowler  113 and Edwards,  i n 1960, found t h a t mice, i n j e c t e d 7 days  mating w i t h 1.0 .yg. o f e s t r a d i o l benzoate  after  or p r o g e s t e r o n e had no 32  implanted embryos on the day of autopsy. attempted  t o h a s t e n ova i m p l a n t a t i o n i n mink by d a i l y i n j e c t i o n s of  a combination benzoate,  o f 2.4 mg. o f p r o g e s t e r o n e and 24 yg. o f e s t r a d i o l  b e g i n n i n g 7 t o 22 days p o s t - c o i t u m .  caused most females kits,  Cochrane and S h a c k e l f o r d  t o be b a r r e n .  The hormonal treatment  I n the animals which d i d produce  the a d m i n i s t r a t i o n of the exogenous e s t r o g e n alone or i n  combination w i t h p r o g e s t e r o n e , r e s u l t e d i n reduced caused  l i t t e r s i z e and  f u r t h e r delay o f o v o - i m p l a n t a t i o n .  The p r o g e s t a t i o n a l p r o l i f e r a t i o n or p r e p a r a t i o n o f the endometrium, n e c e s s a r y f o r i m p l a n t a t i o n of eggs, depends upon a d e l i c a t e 9 between estrogens and p r o g e s t e r o n e .  balance  I t has been adequately r e p o r t e d  t h a t both exogenous estrogens,"'""'"^ and anti-estrogens"'"'"'^ i n t e r f e r e w i t h the f o r m a t i o n of deciduoma p r i o r t o i m p l a n t a t i o n of the f e r t i l i z e d egg. A t p h y s i o l o g i c a l l e v e l s , the estrogens a r e e s s e n t i a l f o r 94 i m p l a n t a t i o n t o occur.  N u t t i n g and Meyer,  i n 1964, r e p o r t e d t h a t the  optimum amount of e s t r o n e r e q u i r e d f o r i n d u c i n g i m p l a n t a t i o n at the normal time appears  t o be between 0.3 and 1.0 yg. d a i l y .  I f the l e v e l of  e s t r o n e i s below a t h r e s h o l d l e v e l a delay i n n i d a t i o n o c c u r s , b u t i f the 94 e s t r o n e l e v e l i s above the t h r e s h o l d l e v e l , n i d a t i o n i s induced. P r o g e s t e r o n e appears t o be v i t a l l y concerned w i t h m a i n t a i n i n g the v i a b i l i t y 88 of the b l a s t o c y s t d u r i n g the extended p r e i m p l a n t a t i o n p e r i o d . Estrogens p l a y an a c t i v e p a r t i n d e t e r m i n i n g whether i m p l a n t a t i o n w i l l occur,  but  only i f r e l a t i v e l y  l a r g e q u a n t i t i e s of p r o g e s t e r o n e a r e  a v a i l a b l e t o ensure the presence of v i a b l e b l a s t o c y s t s . Humphrey, n  88  69 . _ , _. 129 , _ . . 127, 128 xn 1967, Smith and B i g g e r s and Smith, t  i n 1968, have shown t h a t i n the absence of estrogens progesterone implantation w i l l i s primed-lwith  progesterone,  or  n o t occur, but when a uterus  the i n j e c t i o n of s u f f i c i e n t  " 69 (0.024 yg,. e s t r a d i o l , 0.5 yg. 1 7 3  amounts of an e s t r o g e n  -  129 e s t r a d i o l benzoate fertilized  ) will  r e s u l t i n i m p l a n t a t i o n of the  ovum.  Mechanism of A c t i o n I n o r d e r , t o understand the mechanism by which and  antiestrogens  i n t e r f e r e with  estrogens  i m p l a n t a t i o n , the suggested  mechanism of ovum.implantation must f i r s t be d i s c u s s e d .  In  116 1952^  Shelesnyak  reported.that  a n t i h i s t a m i n e s , such as  diphenhydramine or ephedrine h y d r o c h l o r i d e , i n h i b i t e d the formation  of deciduoma when i n t r o d u c e d as a p e l l e t 117  i n t o the u t e r i n e lumen.  L a t e r , Shelesnyak,  or i n s o l u t i o n  i n 1955, and  29 Carlsen, et a l . ,  i n 1961,. a l s o showed that  i n h i b i t e d deciduoma f o r m a t i o n , r e v e r s e d by the c o n c u r r e n t  and t h a t t h i s i n h i b i t i o n was  a d m i n i s t r a t i o n of  These r e s u l t s suggested that a n t i h i s t a m i n e s estrogen/progesterone  decidual c e l l histamine  balance  reaction.  antihistamines  progesterone.  d i s t u r b the c r i t i c a l  f o r the i n d u c t i o n and growth of the 118  I n 1957, Shelesnyak  postulated  that  by some means was r e l e a s e d from the b l a s t o c y s t and  that histamine,  the deciduoma i n d u c i n g f a c t o r , p l u s p r o g e s t e r o n e  o p e r a t i n g on a p r o g r a v i d . uterus  are required f o r s u c c e s s f u l  decidua  -19:  development.  In 1958,  Johnson and  Shelesnyak  73  and  Spazianl  131 and by  Szego  demonstrated t h a t the h i s t a m i n e was  the b l a s t o c y s t , but was  produced by  the uterus  not and  r e l e a s e of the u t e r i n e h i s t a m i n e i s under e s t r o g e n i c 119 120 121 In 1959, estrone  Shelesnyak and  '  '  demonstrated t h a t  that  histamine.  the  control.  estradiol,  e s t r i o l cause the disappearance of.mast c e l l s  the u t e r i n e endometrium thus s u g g e s t i n g  f a c t o r was  produced  from  a reduction i n uterine  Shelesnyak a l s o suggested t h a t a  histamine-releasing-  i n v o l v e d , l i b e r a t e d by  e s t r o g e n s i n the d e c i d u a c e l l 122 reaction. In 1962, Shelesnyak, t h e r e f o r e , suggested t h a t the normal s t i m u l u s f o r i m p l a n t a t i o n i s a surge of estrogens which 79  r e l e a s e h i s t a m i n e from the mast  cells.  123 Shelesnyak, e t . a l .  have shown t h a t an a n t i e s t r o g e n  administered  septematically,  i n h i b i t s n i d a t i o n presumably  antagonizing  the e s t r o g e n surge.  e s t r o g e n or a n t i e s t r o g e n ^ ' " ^  p r o g e s t a t i o n a l s t a t e of the uterus surge of e s t r o g e n and 143 Yochim and  DeFeo  t h u s j a decrease i n i m p l a n t a t i o n  any  an  the  making i t i n s e n s i t i v e t o  the  sites.  144 '  have suggested t h a t n i d a t i o n i s dependent  on f a i r l y narrow l i m i t s of e s t r o g e n and and  by  A l s o , e a r l y i n j e c t i o n of  c o u l d p o s s i b l y antagonize  (MER-25),  change i n the  progesterone  concentration  c i r c u l a t i n g hormone l e v e l would r e s u l t i n 85  the i n t e r r u p t i o n of pregnancy.  Martin  has  shown t h a t  the  h i s t a m i n e - e s t r o g e n r e l a t i o n s h i p i s i n d i c a t i v e t o the u t e r u s , h i s t a m i n e r e l e a s e i s not  i n v o l v e d i n other e s t r o g e n s e n s i t i v e  t i s s u e s , such as those found i n the  vagina.  for  120..  4.  The  e f f e c t s r ; o f e s t r o g e n s and  after implantation  Pregnancy can be implantation  by  estrogens.  i n 1933,  i n t e r r u p t e d a f t e r the completion  ~i i n 1926,  Kelly  of  of e i t h e r e s t r o g e n s or  been noted, however, ever s i n c e the  o n  works of Smith  on pregnancy  occurred.  the . a d m i n i s t r a t i o n  I t has i  has  antiestrogens  anti-  early  IT  o/*  , i n 1931  t h a t as g e s t a t i o n p r o c e e d s , g r e a t e r  and  D'Amour, e t . a l .  q u a n t i t i e s of 47  e s t r o g e n s and  antiestrogens  are r e q u i r e d  to i n t e r r u p t pregnancy  82 Levin, et. a l .  found t h a t e s t r o g e n s a d m i n i s t e r e d  s m a l l doses, over a p e r i o d of 3 days, d u r i n g pregnancy, i n r a t s , d i d not or l e n g t h of  adversely  in relatively  the second h a l f of  a f f e c t e i t h e r the pregnancy  gestation. 99  In 1938,  P a r k e r , e_t. a l .  found t h a t r a b b i t s g i v e n 0.5  or more of e t h i n y l e s t r a d i o l , an o r a l l y a c t i v e e s t r o g e n ,  9 to  days a f t e r mating r e s u l t e d i n r e s o r p t i o n of a l l embryos. r a b b i t s r e c e i v i n g 1.0  mg.  of DES  on  the 13,  found to c o n t a i n 10 embryos, a l l b e i n g a s i m i l a r dose on the 11, 10,  1939,  11 and  12 day  H e c k e l and  12,  i n other 66 Allen  i f administered  seventh day,  of the 32 day  t w e l f t h day establishment  Also,  15 days were  reabsorbed.  Conversely,  13 day  and  h a l f t h i s dose on  r a b b i t s d i d not d i s t u r b pregnancy.  of  the In  estrogen  r e s u l t e d i n r e s o r p t i o n of the r a b b i t  f e t u s e s , but  Huggett and  10  14 and  found t h a t the a d m i n i s t r a t i o n  early a f t e r implantation  occurred.  and  mg.  between the t w e n t i e t h  to twenty-  g e s t a t i o n p e r i o d , spontaneous 68  Pritchard  i n 1945,  abortion  found t h a t a f t e r  of pregnancy, w i t h i n v o l u t i o n of d e c i d u a and of the a l l a n t o i c c i r c u l a t i o n , low  doses of  the  the estronenarid  DMS can no l o n g e r  cause f e t a l death I n r a t s .  Jost  74  i n 1945,  caused r e s o r p t i o n and a b o r t i o n i n mice by the i n j e c t i o n of 5.0 mg. of t e s t o s t e r o n e p r o p r i o n a t e pregnancy, and C o u r r i e r and J o s t  35  on the e i g h t h day o f found t h a t the death of  r a b b i t f e t u s e s o c c u r r e d when 20 mg. of t e s t o s t e r o n e was g i v e n d a i l y  proprionate  t o the mother from 16 t o 21 days of g e s t a t i o n . 137  Velardo,  et. a l . ,  i n 1956, found t h a t e s t r i o l and c e r t a i n of  the C£^-metabolites r e l a t e d t o p r o g e s t e r o n e (e.g. pregnane-3-ok20-£i/cdic and p r e g n a n e d i o n e ) , a d m i n i s t e r e d  i n l a t e r pregnancy t o r a t s ,  r e s u l t e d i n e a r l y a b o r t i o n s , some r e s o r p t i o n o f f e t u s e s and reduced l i t t e r s i z e s ,  Velardo  and h i s c o l l e a g u e s  suggest  that  e a r l y a b o r t i o n can be a s c r i b e d p r i m a r i l y e i t h e r t o inadequacy o f the i m p l a n t a t i o n s i t e or t o a b n o r m a l i t i e s a combination of the two^"^.  of the embryo, or perhaps  I n 1960, Fowler and Edwards"^  demonstrated t h a t mice, when i n j e c t e d w i t h p r o g e s t e r o n e or e s t r a d i o l benzoate i n l a t e pregnancy, r e s u l t e d i n h i g h  fetal  mortality. As w e l l as the animal estrogens estrogens  and a n t i e s t r o g e n s , p l a n t  and a n t i e s t r o g e n s  can r e s u l t i n r e p r o d u c t i v e d i s o r d e r s 84 i n l a t e pregnancy. MacDonald i n 1952, demonstrated t h a t the i n g e s t i o n of p i n e needles or p i n e buds, was the c a u s a t i v e agent of a b o r t i o n and the b i r t h o f weak b e e f c a l v e s .  I n 1964, Cook and  33 Kitts its  a t t r i b u t e d the d e l e t e r i o u s e f f e c t s o f the p i n e needle t o  a n t i e s t r o g e n i c content.  A l l e n and K i t t s  i n 1961, e x t r a c t e d  22; the e s t r o g e n i c components from p i n e needles which were then i n c o r p o r a t e d i n t o a r a t i o n and i m p l a n t a t i o n had  occurred.  had been completely 5..  f e d t o mice a f t e r  theoretical  When a u t o p s i e d , many of the embryos  resorbed.  D i r e c t o e f f e c t s of estrogens  and a n t i e s t r o g e n s on ova and  embryos.  104 I t has been shown t h a t the n a t u r a l estrogens  and  140 progesterone i n e x c e s s i v e amounts are n e i t h e r d i r e c t l y t o x i c to the ova nor are they e s s e n t i a l f o r normal cleavage up to the 25 b l a s t u l a stage.  Burdick, et. a l . ,  found t h a t a f t e r  estrogen  i n j e c t i o n , the normal round b l a s t o c y s t s t a g e , i n which the trophoblast c e l l s  are i n c o n t a c t w i t h the zona p e l l u c i d a , i s 3 4  u s u a l l y a t t a i n e d by the d e v e l o p i n g t u b a l ova. concluded  In 1942,  Alden  '  t h a t no s p e c i a l u t e r i n e environment i s n e c e s s a r y f o r  p r o d u c t i o n of the b l a s t o c y s t , nor are o v a r i a n s e c r e t i o n s necessary f o r the e a r l y stages of the b l a s t o c y s t  formation.  I n f a c t , mouse  eggs have shown development when p l a c e d i n t o the a n t e r i o r of the  chamber  eye.  A n t i m e t a b o l i t e s , however, such as 6 d i a z o 5 oxo L - n o r l e u c i n e , 9 and s y n t h e t i c estrogens and a n t i e s t r o g e n s , such as DES and M E R - 2 5 a r e c y t o t o x i c t o the d e v e l o p i n g ova. Even a f t e r 39 i m p l a n t a t i o n , compounds such as n o r e t h y n o d r e l induce extence effects  fetal destruction.  of the estrogens  and  It is felt  and others  can  that c y t o t o x i c  a n t i e s t r o g e n s , however, i s not due 76 t h e i r r e s p e c t i v e hormonal a c t i v i t i e s p e r se.  to  23.  I l l M a t e r i a l s and Methods PART A:  Feeding  Trial  A study e x t e n d i n g from A p r i l 3, 1967 to May 19, 1967 was designed to observe the e f f e c t s of d a i l y f e e d i n g of DES and a f o r a g e a n t i e s t r o g e n e x t r a c t on female mink d u r i n g pregnancy.  The two hormones  were not mixed i n t o the d a i l y ranch r a t i o n . u n t i l i m p l a n t a t i o n of the fertilized  egg was assumed t o have o c c u r r e d i n the female.  Commercial  powdered DES was used, w h i l e the a n t i e s t r o g e n was e x t r a c t e d from ground  a l f a l f a leaf-meal.  dehydrated  DES was i n c o r p o r a t e d i n t o the mink r a t i o n , so  t h a t the animals r e c e i v e d 5, 10, or 15 meg. per day.  The a n t i e s t r o g e n  e x t r a c t was i n c o r p o r a t e d i n t o the d i e t so t h a t the mink consumed 5, 10, or 15, grams e q u i v a l e n t of a l f a l f a p e r day. 1.  Materials a.  Into a c o m p l e t e l y randomized  mink were d i s t r i b u t e d b.  Housing  design, t h i r t y standard  female  i n t o 6 groups w i t h 5 animals per group.  - A l l animals were housed a t the U.B.C. mink u n i t i n  i n d i v i d u a l w i r e cages  (17" x 15" x 1 5 " ) .  A wooden next box (8.5" x 7" x  7.5") was a t t a c h e d to one end of the cage and straw was used as bedding. 2.  Methods a.  day.  F e e d i n g - The 6 e x p e r i m e n t a l r a t i o n s were mixed once every  second  S u f f i c i e n t amounts of each r a t i o n were mixed and weighed i n t o e q u a l  p o r t i o n s t o permit d a i l y f e e d i n g of the r a t i o n s .  The second day's f e e d  was kept r e f r i g e r a t e d u n t i l f e d . To reduce the p o s s i b i l i t y of DES or a n t i e s t r o g e n c o n t a m i n a t i o n i n the f e e d f o r the remainder  of the U.B.C. herd  24.  (the c o n t r o l g r o u p s ) , a l l mixing was to where the c o n t r o l f e e d was  done a t a s e p a r a t e l o c a t i o n  mixed.  feed on top o f the e x p e r i m e n t a l cages  A wooden cover was  p l a c e d over  to prevent f u r t h e r  contamination  by p h y s i c a l t r a n s f e r of feed to the s u r r o u n d i n g cages. ad  distant  Water was  the  fed  libitum. b.  E x t r a c t i o n of a n t i e s t r o g e n - The e x t r a c t i o n of the  alfalfa g  a n t i e s t r o g e n was  a m o d i f i c a t i o n of the method developed by L u i z z o , et^. a l .  (See Appendix ,1).  A l l a l f a l f a e x t r a c t i o n s were s t o r e d under  u n t i l mixed i n t o the ranch c.  was  ration.  A n t i e s t r o g e n a c t i v i t y - The hormonal a c t i v i t y of the a n t i e s t r o g e n g  determined d.  week.  refrigeration  u s i n g the Astwood, s i x - h o u r b i o a s s a y .  O r a l a c t i v i t y of DES  - DES  was  (See Appendix I I )  f e d to immature female r a t s f o r one  At the end of t h i s p e r i o d the animals were s a c r i f i c e d and  were d i s s e c t e d f r e e , trimmed of f a t and weighed. expressed as a percentage  of body  S t a t i s t i c a l A n a l y s i s ; - Treatment 132. A n a l y s i s of V a r i a n c e PART B: Feeding T r i a l  e f f e c t s of DES u n t i l May  a second  e f f e c t s were a n a l y s e d u s i n g the  experiment  was  f e d a t v a r i o u s stages of pregnancy.  11, 1968.  The u t e r i n e response i s  weight.  e.  On March 4, 1968  the u t e r i  F i f t e e n mcgms. of DES  mink a c c o r d i n g to the f o l l o w i n g scheme:  was  initiated  to study  The experiment  f e d to 6 groups of  the  extended female  25.  Group I  F o r one week  pretupping.  Group I I  From b r e e d i n g  to implantation.  Group I I I From i m p l a n t a t i o n  and  t o the end o f g e s t a t i o n .  Group IV  From one week p r e t u p p i n g  Group V  From b r e e d i n g  Group VI  From one week p r e t u p p i n g  to implantation.  t o the end o f g e s t a t i o n , t o the end o f g e s t a t i o n .  The a l f a l f a a n t i e s t r o g e n e x t r a c t was f e d t o 3 groups of mink a f t e r i m p l a n t a t i o n had o c c u r r e d  a t l e v e l s as d e s c r i b e d  i n Part A of t h i s  study. 1.  Materials a.  Animals - I n the DES study, 70 p a s t e l female mink were d i s t r i b u t e d  randomely i n t o 7 groups w i t h  10 animals p e r group.  no DES.  study, 20 p a s t e l mink were randomized  I n the a n t i e s t r o g e n  4 groups w i t h  5 animals p e r group,  The c o n t r o l s r e c e i v e d into  One group, the c o n t r o l s , r e c e i v e d no  antiestrogen. b.  Housing - A l l animals were housed as d e s c r i b e d  i n Part A of t h i s  study. 2.  Methods a.  F e e d i n g - A l l m i x i n g and f e e d i n g o f the e x p e r i m e n t a l  performed a c c o r d i n g  t o the procedure d e s c r i b e d  r a t i o n s were  i n Part A of t h i s  b.  E x t r a c t i o n of a n t i e s t r o g e n - See p a r t A of t h i s  c.  Antiestrogen  d.  S t a t i s t i c a l A n a l y s i s - See P a r t A o f t h i s  study.  and DES a c t i v i t y - See P a r t A of t h i s study.  study.  study."  IV  Results  and  The  Discussion  e s t r o g e n i c a c t i v i t y of the DES  and  the a n t i e s t r o g e n i c  a c t i v i t y of the a l f a l f a e x t r a c t used i n P a r t A and tested before  each experiment was  a l f a l f a e x t r a c t bioassays  initiated.  are g i v e n  (Tables  The  B of Study I were r e s u l t s of  I and  II).  The  the uterine  response to the hormonal i n j e c t i o n s i s expressed as a percentage of body weight to a d j u s t f o r the v a r i a t i o n s i n body s i z e . by means of an A n a l y s i s of V a r i a n c e ,  I t was  t h a t the d i f f e r e n c e s between  average u t e r i n e weights i n the t h r e e treatments of the f i r s t (Table I) were s i g n i f i c a n t bioassay  ( P ^ 0.025).  The  c o n t r o l group.  a n t i f e r t i l i t y agent.  was  bioassay  e x t r a c t was  o r a l a c t i v i t y of the DES  done.  The  was  only  also tested.  i n Table  I and  The  II,  same source  I I I show t h a t  to immature, o v a r i e c t o m i z e d  (P< 0.05)  an  significantly antiestrogenic.  Wistar  i n c r e a s e i n u t e r i n e weight.  range s t u d i e d , each i n c r e a s e i n the l e v e l of DES significant  the  dehydration  B of t h i s study, t h e r e f o r e o n l y  r e s u l t s presented  o r a l a d m i n i s t r a t i o n of DES caused a s i g n i f i c a n t  , but caused a  From the r e s u l t s , t h e r e f o r e , i n Tables  used f o r P a r t s A and  was  lower than  be of some s i g n i f i c a n c e i n i t s a c t i o n as  can be s a i d t h a t the a l f a l f a  of DES  percent  T h i s suggests t h a t the a l f a l f a a n t i e s t r o g e n not  of the u t e r u s , which may  The  weight as a  + C.E.C.) was  a c t e d a n t a g o n i s t i c a l l y to the e s t r a d i o l 17-3  it  bioassay  to a h i g h e r degree (P< 0.005).  f o r P a r t A the average u t e r i n e  body weight i n group I I I ( e s t r a d i o l - 1 7 3  the  r e s u l t s of the second  (Part B) were a l s o s i g n i f i c a n t , but  In the b i o a s s a y  found,  (P <0.05) i n c r e a s e i n u t e r i n e weight.  one the  rats  Over  the  fed r e s u l t e d i n a A p l o t of the  data  ( F i g . I) shows a t y p i c a l l i n e a r dose/response r e l a t i o n s h i p between the  TABLE I Antiestrogenic  Body Weight (gms)  Group I  A c t i v i t y o f the A l f a l f a  0.025  0.025  32.8 28.1 36.0 26.5 30.1  y g E s t r a d i o l 17-3 77.4 68.1 83.6 90.3 83.2  a  21.1 23.8 22.0 27.7 32.4  yg E s t r a d i o l 17-3 77.4 85.6 82.7 85.8 72.5  III  Uterine Weight (% body wt.)  C o n t r o l s 0.2 ml. p h y s i o l o g i c a l 79.1 82.4 70.4 78.5 80.3  II  Uterine Weight (mgm)  S . E . = Standard  20.4 16.9 23.5 22.7 26.0  Error  E x t r a c t Used i n Study I P a r t A. Average U t e r i n e Weight * S.E. (% body wt.)  s a l i n e (0.9% s a l i n e )  0.0267 0.0289 0.0313 0.0353 0.0403  a  per r a t .  0.0325 ± 0.0026  i n 0.2 ml 0.9% s a l i n e per r a t . 0.0424 0.0328 0.0435 0.0309 0.0415  0.0382 - 0.0026  + CEC (10 gm D.M./rat) i n 0.2 ml 0.9% s a l i n e 0.0264 0.0248 0.0281 0.0251 0.0313  0.0271 - 0.0019  TABLE I I  A n t i e s t r o g e n i c A c t i v i t y o f the A l f a l f a E x t r a c t used i n Study I P a r t B.  Body Weight (gm)  Group I  0.025  0.025  0.0368 0.0264 0.0369 0.0307 0.0385 0.0228  32.0 34.0 25.0 24.0 34.0 32.5  Average U t e r i n e Wt. - S.E. (% Body Wt.) (0.9% s a l i n e )  0.0320 - 0.0026  in 0  0.0502 0.0498 0.0359 0.0447 0.0466 0.0436  0.0451 - 0.0021  y g E s t r a d i o l 17-- B + c :  59.9 63.0 52.2 51.1 50.2 47.1  ;  22.0 16.0 25.0 23.0 22.0 14.0  y g E s t r a d i o l 17- 6  63.7 68.3 69.7 53.7 73.0 74.5 III  Uterine Weight (% Body Wt.)  C o n t r o l s 0.2 ml p h y s i o l o g i c a l s a l i n e 59.8 60.6 67.8 75.0 57.2 61.5  II  Uterine Weight (mgm)  22.0 19.0 21.0 16.5 15.0 15.5  Standard E r r o r  0.0367 0.0302 0.0402 0.0323 0.0299 0.0329  0.0337 - 0.0016  TABLE I I I  The E s t r o g e n i c A c t i v i t y o f DES A d m i n i s t e r e d W i s t a r Rats.  Group I  Body Weight (gm) Controls: 57.3 68.2 71.4 72.0 53.3 68.1  II  Average U t e r i n e Wt. - S.E. (% Body Wt.)  0 mcgm DES/rat/week. 25.6 33.8 18.9 23.0 20.8 19.1  0.0447 0.0496 0.0265 0.0319 0.0390 0.0280  0.0366 - 0.0038  30.2 22.1 28.5 18.4 40.8 23.8  0.0419 0.0332 0.0544 0.0292 0.0609 0.0408  0.0434 - 0.0049  0.300 mcgm DES/rat/week 67.3 60.1 60.4 64.6 56.8 70.2  IV  Uterine Weight (% Body  0.150 mcgm DES/rat/week 72.1 66.6 52.4 63.0 67.0 58.4  III  Uterine Weight (mgm)  O r a l l y to Weanling Female  0.350 61.7 52.0 61.6 71.5 . 62.4 59.0  = Standard  43.0 38.1 27.9 35.9 30.9 33.1  0.0639 0.0634 0.0462 0.0556 0.0544 0.0472  0.0551 - 0.0031  DES/rat/week 45.8 38.5 28.8 40.0 46.2 21.5  Error  0.0742 0.0740 0.0468 0.0559 0.0740 0.0364  0.0602 - 0.0067  a  30.  31.  l e v e l of DES  f e d and the u t e r i n e response.  The response can only  be  s a i d to be l i n e a r w i t h i n the range s t u d i e d , because f u r t h e r  increases  i n the l e v e l of DES  relation-  f e d would  e v e n t u a l l y show a c u r v i l i n e a r  s h i p as a maximal u t e r i n e response was  approached .  In P a r t A of t h i s study the DES  and a n t i e s t r o g e n were not  f e d to the mink u n t i l i m p l a n t a t i o n had o c c u r r e d .  The l e n g t h of g e s t a t i o n  i n the mink v a r i e s from 42 t o 79 days w i t h an average of 51 days  38  54 The embryos implant about 28 t o 30 days b e f o r e p a r t u r i t i o n and a n t i e s t r o g e n ,  t h e r e f o r e , were not i n c o r p o r a t e d  d i e t u n t i l 30 days a f t e r c o i t u s . occurred  I t was  .  IV).  assumed t h a t i m p l a n t a t i o n  had  i n a l l a n i m a l s by t h i s time.  or the a n t i e s t r o g e n was  incorporated  The l e v e l s of 5, 10, and 15 meg.  a t l e v e l s of 5, 10, and 15 gm.  DES  equivalent  to produce a complete r e p r o d u c t i v e  i n t o the d i e t  or the p l a n t  occurred  (see T a b l e  antiestrogen  of a l f a l f a were not  f a i l u r e i n any one group.  group, however, some females were b a r r e n period.  DES  i n t o the mink's  A d e f i n i t e d e c r e a s e i n the number o f k i t s per female when DES  The  sufficient In each  at the end of the g e s t a t i o n  I t has been demonstrated many times t h a t as g e s t a t i o n proceeds  g r e a t e r q u a n t i t i e s of e s t r o g e n i c and a n t i e s t r o g e n i c compounds a r e required  to i n t e r r u p t pregnancy and f o r some a n i m a l s i n t h i s study the  l e v e l s of hormones used were not s u f f i c i e n t tive failures.  to cause complete r e p r o d u c -  Other f a c t o r s which would c o n t r i b u t e to v a r i a t i o n s i n  the s e v e r i t y of hormonal a d m i n i s t r a t i o n ,  include:  (1) i n d i v i d u a l s e n s i t i v i t y to hormonal a c t i o n s , (2) v a r i a t i o n s i n the stage of g e s t a t i o n , and  (3) v a r i a t i o n s i n feed i n t a k e which would  a v a r i a t i o n i n the l e v e l of hormonal  intake.  cause  32.  TABLE IV  R e p r o d u c t i v e Performance of Mink R e c e i v i n g DES or A n t i e s t r o g e n Ova I m p l a n t a t i o n - P a r t A  after  DES L e v e l (meg/mink/day)  No. of Females Mated  No. of Litters Born  Total Kits Born  5  5  3  7  II  10  5  2  8  III  15  5  4  15  5  3  15  3.0  5  3  14  2.8  5  2  8  1.6  169  154  612  3.6  Group I  No. Per  Kits  Female 1.4 1.6 3.0  Antiestrogen Level-'(gm/mink/day) I  5-  II III Control  10 15 0  Antiestrogen extract incorporated grams of a l f a l f a  i n e q u i v a l e n t of  33. - 3 -  In 1960, T r a v i s  136  , i n a s i m i l a r study  o f DES f e e d i n g , found  almost complete f a i l u r e of the normal r e p r o d u c t i v e p r o c e s s e s . as a lowered k i t p r o d u c t i o n ,  As w e l l  T r a v i s found r e a b s o r p t i o n o f l i t t e r s ,  lower k i t weights and g r e a t e r k i t m o r t a l i t y i n mink r e c e i v i n g t h e DES. 136 Spontaneous a b o r t i o n was n e i t h e r r e p o r t e d by T r a v i s i n the p r e s e n t  nor observed  study.  P a r t B of t h i s study was i n i t i a t e d season (March, 1968). of the p r e v i o u s  the f o l l o w i n g b r e e d i n g  I t was thought p o s s i b l e , a f t e r s t u d y i n g  experiment  the data  (Part A ) , t h a t a dose/response r e l a t i o n s h i p  c o u l d e x i s t between the l e v e l of hormone a d m i n i s t e r e d No such r e l a t i o n s h i p was e v i d e n t  and k i t p r o d u c t i o n .  i n t h e DES groups o f P a r t A, but t h e r e  i s a p o s s i b i l i t y o f such a r e l a t i o n s h i p i n the a n t i e s t r o g e n groups. The  a n t i e s t r o g e n experiment was, t h e r e f o r e , repeated  the f o l l o w i n g  breeding  season and to g a i n f u r t h e r knowledge of the e f f e c t s of DES on r e p r o d u c t i o n , 15 meg. o f DES was f e d a t v a r i o u s stages  o f pregnancy.  The  r e s u l t s of P a r t B a r e g i v e n i n T a b l e V. The  f e e d i n g of DES d u r i n g any of t h e v a r i o u s stages of pregnancy  d e s c r i b e d was c a t a s t r o p h i c (Table V ) . ment, one female d i e d b e f o r e  In Group I I I of the DES e x p e r i -  the completion  o f the study.  6 f e t u s e s were found i n u t e r o , w i t h 2 f e t u s e s p a r t i a l l y  Upon necropsy,  reabsorbed.  None of the o t h e r animals were s a c r i f i c e d f o r necropsy. Feeding prevents  DES one week b e f o r e b r e e d i n g  o v u l a t i o n by c h e m i c a l l y  antagonizing  (Group I , IV, and VI) the s e c r e t i o n o f the  72 gonadotrophins . The i n h i b i t i o n o f t h e gonadotrophins prevents " 112 oogenesis and subsequent o v u l a t i o n of the ovum . When DES i s f e d immediately a f t e r mating  (Groups I I and V the hormone d i s r u p t s the  34. - 4 -  t r a n s p o r t of t h e ova through t h e F a l l o p i a n tubes.  I t has been r e c e n t l y  suggested by Humphrey^, t h a t low doses of an e s t r o g e n t u b e - l o c k i n g of t h e ova a t t h e i s t h m o - u t e r i n e  o r ampulla-isthmus  t i o n as w e l l as a c c e l e r a t i o n o f t h e t u b a l ova. reach t h e u t e r u s n o r m a l l y ,  can cause both  I f the f e r t i l i z e d ova  t h e f e e d i n g of DES a t t h i s time would d i s r u p t  the hormonal c o n c e r t a s s o c i a t e d w i t h pregnancy and prevent I f DES i s n o t f e d u n t i l  junc-  t h e ova a r e implanted  implantation.  (Group I I I ) , r e a b s o r p t i o n  or spontaneous a b o r t i o n of t h e f e t u s e s may r e s u l t . In t h e a n t i e s t r o g e n study  of P a r t B, i t can be seen from t h e  r e s u l t s i n T a b l e V, t h a t a r e d u c t i o n i n p r o d u c t i v i t y o c c u r r e d when t h e a n t i e s t r o g e n was i n c o r p o r a t e d i n t o t h e r a t i o n .  The r e s u l t s , however,  suggest t h a t no dose/response r e l a t i o n s h i p e x i s t s between t h e l e v e l of a n t i e s t r o g e n f e d and the number of k i t s born p e r female.  I n 1962,  53 Emmens and F i n n  c o u l d n o t f i n d any dose/response r e l a t i o n s h i p between  progesterone or ethyl-19-nortestosterone  and l i t t e r  s i z e by e i t h e r  l o c a l o r p a r e n t e r a l a d m i n i s t r a t i o n i n pregnant r a t s and mice. From the r e s u l t s of Study I i t i s e v i d e n t  t h a t DES a t l e v e l s  as low as 5 t o 15 mcgm /mink/day can cause complete r e p r o d u c t i v e At t h e 15 mcgm. l e v e l i t i s p o s s i b l e to prevent if  t h e hormone i s a d m i n i s t e r e d  breeding  o r d i s r u p t pregnancy  f o r a t l e a s t one week from one week b e f o r e  t o 30 days b e f o r e p a r t u r i t i o n .  The f o r a g e a n t i e s t r o g e n c a n  a l s o reduce t h e p r o d u c t i v i t y o f the mink even when fed a f t e r t i o n has o c c u r r e d . agent as i s DES. estrogen,  failure.  implanta-  I t i s not as e f f e c t i v e , however, as an a n t i f e r t i l i t y The present  concentrated  experiments show t h a t the a l f a l f a  i n t o an e x t r a c t , i s capable  anti-  of i n t e r r u p t i n g  pregnancy, however i t s c o n c e n t r a t i o n i n the p l a n t i t s e l f may be so low  TABLE V  R e p r o d u c t i v e Performance of Mink R e c e i v i n g DES Pregnancy or A n t i e s t r o g e n a f t e r I m p l a n t a t i o n  at V a r i o u s  Stages of  DES L e v e l (meg/mink/day)  No. of Females Mated  No. of Litters Born  Total Kits Born  No. of Kits p Female  I  15  10  l  1  0.1  II  15  10  1  1  0.1  III  15  10  l  4  0.4  IV  15  10  1  1  0.1  V  15  10  0  0  0  VI  15  10  0  0  0  0  10  7  49  4.9  5  5  3  15  3.0  II  10  5  5  18  3.6  III  15  5  4  21  4.2  0  5  5  29  5.8  Group  Control  a  b  Antiestrogen Level (gm/mink/day) c  I  Control  One  k i t b o r n dead  'One animal i n Group I I I d i e d and were found i n u t e r o Antiestrogen extract incorporated g r a i n s of a l f a l f a  upon necropsy, 6 f e t u s e s  i n equivalents  of  that  the  i s of no  antiestrogen level significance.  i n a mink r a t i o n from n a t u r a l  sources  37.  V  Conclusions  1.  The d a i l y f e e d i n g o f 5, 1G o r 15 meg. o f DES f o r one week, t o  female mink, a t any stage o f the r e p r o d u c t i v e p r o c e s s breeding  t o immediately a f t e r i m p l a n t a t i o n )  (from one week b e f o r e  can cause complete  reproductive  failure. 2.  The f e e d i n g of a f o r a g e  ( a l f a l f a ) antiestrogen extract at l e v e l s  e q u i v a l e n t t o 5, 10 or 15 gm. of a l f a l f a has  t o pregnant mink a f t e r  o c c u r r e d w i l l r e s u l t i n a fewer number of k i t s per l i t t e r  not r e c e i v i n g 3.  implantation  than mink  the a n t i e s t r o g e n e x t r a c t .  The f e e d i n g o f v a r i o u s l e v e l s o f DES o r the a n t i e s t r o g e n e x t r a c t  does n o t adhere t o a dose/response r e l a t i o n s h i p e i t h e r i n i n t e r r u p t i n g pregnancy o r r e d u c i n g 4.  the number of k i t s per female.  DES, f e d a t l e v e l s of 5, 10 o r 15 mcgm. per mink per day i s a  more p o t e n t l a h t i f e r t i l i t y agent than the f o r a g e a n t i e s t r o g e n f e d a t l e v e l s e q u i v a l e n t t o 5, 10 or 15 gm. o f a l f a l f a .  STUDY I I  The E f f e c t s of V a r i o u s P r o t e i n and Energy L e v e l s on-the Maintenance and E a r l y Growth of Mink, (Mustela v i s o n ) . PART A.  Maintenance  PART B.  E a r l y Growth  38.  I.  Introduction:  Among the v a r i o u s n u t r i t i o n a l i n t e r r e l a t i o n s h i p s a f f e c t i n g the dynamic s t a t e of the complex organism, the r e l a t i o n s h i p between p r o t e i n metabolism and investigated. Firstly,  energy has  P r o t e i n has  two  been the most  f u n c t i o n s w i t h i n the animal body.  i t i s e s s e n t i a l f o r the s y n t h e s i s  s e c o n d l y , i t i s a source of energy. t h a t i f the amino a c i d c o n s t i t u e n t s tissue synthesis,  they are not  and  i f they are s u p p l y i n g 101 synthesis.  extensively  of body t i s s u e s  However, i t must be  and remembered  of the f e d p r o t e i n are used f o r  supplying  energy f o r m e t a b o l i c  energy, they are not  processes;  available for tissue  I t i s w e l l e s t a b l i s h e d t h a t the d i e t a r y energy l e v e l has  a  124 profound e f f e c t on food t h a t 74%  consumption.  of the v a r i a t i o n i n food  S i b b a l d , et a l ,  have shown  consumption i n r a t s i s a s s o c i a t e d  w i t h the apparent d i g e s t i b l e energy content of the r a t i o n .  Within  p h y s i o l o g i c a l l i m i t s , the need to s a t i s f y energy requirements 89 provides  the primary s t i m u l u s  to food  intake.  evidently  I t i s understandable,  t h e r e f o r e , t h a t the d i e t a r y requirements f o r n u t r i e n t s and  the  efficiency  of n u t r i e n t u t i l i z a t i o n are c l o s e l y r e l a t e d to the i n t a k e of energy. The  primary need of the body i s f o r energy, and  its priority  over  the need f o r p r o t e i n s y n t h e s i s should be assumed, s i n c e s y n t h e s i s i s an 101 . energy r e q u i r i n g p r o c e s s . The primary f u n c t i o n of p r o t e i n , t i s s u e s y n t h e s i s , can take p l a c e only i f the energy needs of the organism are •A  A  provided  *  for.  101  39.  L i t e r a t u r e Review  A.  R e l a t i o n s h i p of p r o t e i n u t i l i z a t i o n to energy 1)  The e f f e c t s of constant energy i n t a k e .  intake.  p r o t e i n l e v e l s with  varying  P r o t e i n u t i l i z a t i o n i n an a d u l t i n d i v i d u a l on adequate p r o t e i n i n t a k e and  nitrogen equilibrium w i l l  an  respond  101 to changes i n the energy content  of the d i e t .  From s t u d i e s  on u n d e r n u t r i t i o n i t i s apparent that w i t h d r a w a l of energy i n the form of e i t h e r carbohydrate or f a t r e s u l t s i n a decrease i n . 18 ' . . ^ , 90 nitrogen retention e v i d e n t by an impaired n i t r o g e n b a l a n c e . 3  Animals r e s t r i c t e d fall  i n energy i n t a k e below a maintenance  i n t o a negative  n i t r o g e n b a l a n c e and  level  the degree of  n e g a t i v i t y i s d i r e c t l y r e l a t e d to the s e v e r i t y of the  caloric  27 restriction.  With s u f f i c i e n t  f a t and  l a b i l e protein stores,  the i n c r e a s e i n n i t r o g e n e x c r e t i o n on a c a l o r i c d e f i c i e n t  diet  is  reserves  a r e s u l t of the animal c a t a b o l i z i n g i t s l a b i l e p r o t e i n  109 to p r o v i d e  s u f f i c i e n t energy f o r the e s s e n t i a l a n a b o l i c  During the i n i t i a l  stages  of c a l o r i c r e s t r i c t i o n ,  the  functions.  nitrogen  b a l a n c e index, which i s a a f u n c t i o n of the n i t r o g e n r e t a i n e d i n the body of the animal and  i s d e f i n e d as the r a t e of change of 107  n i t r o g e n balance with If  respect  to absorbed n i t r o g e n , i s unchanged.  the degree of c a l o r i c d e p l e t i o n i s not  r a p i d l o s s of body n i t r o g e n t a p e r s  too marked, the  initial  o f f , which i n d i c a t e s t h a t  animals are capable o f a a d a p t i n g to c a l o r i c r e s t r i c t i o n s by r e d u c i n g 89 19 their catabolic activity. In the growing animal, Bosshardt estimated t h a t a 34 percent decrease i n energy i n t a k e r e s u l t s i n a 14 percent decrease i n energy e x p e n d i t u r e . T h i s shows t h a t  40-.-  i n t h e growing animal on a constant conservation  p r o t e i n i n t a k e energy  i s accomplished by a r e d u c t i o n i n b a s a l  19 metabolism.  I f the c a l o r i c  restriction  the animal becomes s e v e r e l y d e p l e t e d  i s marked, however,  i n f a t and t i s s u e p r o t e i n 109  reserves  and the n i t r o g e n b a l a n c e index i s reduced  catabolism  o f t i s s u e s i s again  and the  increased eventually  culminating  DO  i n death o f t h e animal. The  r e s i s t a n c e to c a l o r i c  part, with reserves  restriction i s correlated, i n  the magnitude of t h e p r o t e i n s t o r e s and the c a l o r i c  of the b o d y . ^ ^  R o s e n t h a l and A l l i s o n " ' " ^ found t h a t  t h e r e were d i f f e r e n t i a l changes i n body t i s s u e s i n animals on caloric deficient  diets.  T h i s demonstrated t h a t some organs,  such as the l i v e r , were more l a b i l e to n e u t r a l l i p i d d e p l e t i o n than other  organs, such as the h e a r t .  therefore, to a c a l o r i c  and p r o t e i n  The response,  r e s t r i c t i o n i s a f u n c t i o n o f the p h y s i o l o 109  g i c a l s t a t e of t h e animal as w e l l as the n a t u r e of the d i e t . Energy has a s p a r i n g e f f e c t intake increases with increases  on p r o t e i n n e t a b o l i s m .  p r o t e i n intake constant,  As c a l o r i c  protein u t i l i z a t i o n  t o a maximum, beyond which no a d d i t i o n a l p r o t e i n  u t i l i z a t i o n occurs."'"^"'" young e s s e n t i a l l y  Calloway and S p e c t o r ^  found t h a t f o r  normal a c t i v e men, i n n e g a t i v e  nitrogen  balance,  when no p r o t e i n i s f e d , t h e p r o t e i n d e f i c i t - can be maximally reduced by s u p p l y i n g  about 700 n o n - p r o t e i n  p r o t e i n - s p a r i n g i s achieved 26 absence o f p r o t e i n .  calories.  No  significant  by i n t a k e s as h i g h as 2000 K c a l i n the  When the d i e t  provides  adequate amounts  of p r o t e i n , increments i n energy i n t a k e , produced by adding c a r b o h y d r a t e or f a t t o a sub-maintenance d i e t ,  causes a l i n e a r  41.  improvement i n n i t r o g e n b a l a n c e through e q u i l i b r i u m to a considerable thus be  nitrogen retention.  92  P r o t e i n m e t a b o l i s m must  i n a s t a t e of dynamic e q u i l i b r i u m w i t h  energy i n t a k e , 92  even when the animal i s i n n i t r o g e n e q u i l i b r i u m .  Munro  and  92 Naismith  a l s o found t h a t the t o t a l amount of p r o t e i n  contained  i n the l i v e r responded to i n c r e a s i n g energy i n t a k e i n a fashion.  Changes i n energy i n t a k e caused a s m a l l e r  change i n the n i t r o g e n content 92 • content  of the  of the c a r c a s s  linear  percentage  than i n the  nitrogen  liver.  Once a c e r t a i n energy i n t a k e has diet a d d i t i o n a l increases  been reached on a p r o t e i n - f r e e  i n energy i n t a k e w i l l not 92  r e d u c t i o n i n n i t r o g e n output.  T h i s means t h a t when the supply  amino a c i d s c i r c u l a t i n g to the t i s s u e s comes s o l e l y endogenous s o u r c e s ,  l e a d to f u r t h e r  from  t h i s becomes a l i m i t i n g f a c t o r i n the  of p r o t e i n s y n t h e s i s at q u i t e low  of  l e v e l s of energy i n t a k e .  rate 92  When the d i e t s u p p l i e s adequate p r o t e i n t h i s l i m i t a t i o n i s no 92 longer present. However, the major f a c t o r l i m i t i n g growth through out a wide range of c a l o r i c i n t a k e s i s s t i l l the amount 19 102 of p r o t e i n i n g e s t e d . During pregnancy, P i k e , et a l have shown t h a t i n the r a t the d u r i n g organogenesis was  l i m i t i n g f a c t o r of n i t r o g e n r e t e n t i o n the supply  of n o n - p r o t e i n  than the l e v e l of p r o t e i n .  However, d u r i n g  f e t a l growth c a l o r i e s are no  longer  retention;  calories,  rather  the p e r i o d of r a p i d  the f a c t o r l i m i t i n g  nitrogen  r a t h e r , i t i s the l e v e l of n i t r o g e n i n the d i e t  that  1  n? i s imposing the l i m i t a t i o n . I t i s p o s s i b l e t h a t t h e r e is."a: c a l o r i c i n t a k e f o r each p r o t e i n i n t a k e , the p e r f e c t b a l a n c e between p r o t e i n and 109 mass.  c a l o r i e s r e s u l t i n g i n an adequate development of body  42.  2)  The e f f e c t s of constant protein intake.  energy l e v e l s w i t h  varying  •Although the e f f i c i e n c y of p r o t e i n u t i l i z a t i o n ,  in  c o n d i t i o n s of c a l o r i e u n d e r n u t r i t i o n , i s governed l a r g e l y by the extent  of the c a l o r i c r e s t r i c t i o n ;  amounts of p r o t e i n can be u t i l i z e d by intake i s increased while  within limits,  increasing  the body i f the p r o t e i n  the n o n - p r o t e i n  caloric  content  of  19 the r a t i o n i s kept c o n s t a n t . c a l o r i c intake constant, a peak r a t e and Rosenthal''"^'  7  As  the p r o t e i n i n c r e a s e s  protein u t i l i z a t i o n r i s e s sharply  the r i s e becomes p r o g r e s s i v e l y  found t h a t dogs f e d r e s t r i c t e d d i e t s u t i l i z e d  low.  was  Additional dietary protein resulted i n a  s m a l l p o s i t i v e n i t r o g e n b a l a n c e which c o u l d not be f u r t h e r no matter how The  to  less.^^  n i t r o g e n i n a normal f a s h i o n when the p r o t e i n content relatively  with  increased  much p r o t e i n i s i n c l u d e d i n the  net p r o t e i n u t i l i z a t i o n , however, decreases  diet."^^ linearly  as the percentage of p r o t e i n c a l o r i e s i n the d i e t i s i n c r e a s e d , 89 and the r a t e of decrease i s a c h a r a c t e r i s t i c of each protexn. 28 Calloway and  Spector  rats diminished  have shown t h a t the n i t r o g e n u t i l i z a t i o n i n  from a p p r o x i m a t e l y 55  l e v e l of d i e t a r y n i t r o g e n i n c r e a s e d Thus i n d i c a t i n g an i n c r e a s e d use  to 17 percent  from 75 to 604  when the mg.  daily.  of d i e t a r y p r o t e i n f o r energy.  43-.  B.  The e f f e c t s of carbohydrate and f a t on p r o t e i n  Although the c a l o r i c i n t a k e protein u t i l i z a t i o n , to f a t f e e d i n g  exerts  a s i g n i f i c a n t e f f e c t on  the d i f f e r e n c e i n response to carbohydrate  c l e a r l y i n d i c a t e s t h a t carbohydrate a l s o e x e r t s  e f f e c t s e p a r a t e from i t s c a l o r i g e n i c function."^"*" are adequate  utilization.  i n p r o t e i n and energy, replacement  " c a l o r i e s by f a t c a l o r i e s produces e x c r e t i o n which then d e c l i n e s .  On d i e t s  Furthermore,  19  Munro and N a i s m i t h  protein-free diet, the addition ;  from  900  to 1700  Kcal/m  2  92  i n complete of  have shown t h a t i n r a t s on a  to i n f l u e n c e n i t r o g e n  a d d i t i o n of carbohydrate produced  protein  sparing  of f a t t o b r i n g the energy  failed  that  i n nitrogen  s t a r v a t i o n c a r b o h y d r a t e b u t not f a t , has the p r o p e r t y body p r o t e i n .  an  of carbohydrate  a transient increase  101  and  intake  balance.  up The  2 Kcal/m. ,  some improvement up t o 1200  92 but not t h e r e a f t e r . time of f e e d i n g  In the case of the r a t , the s e p a r a t i o n  d i e t a r y p r o t e i n and carbohydrate  of the  (but not f a t ) has  90 an adverse e f f e c t on n i t r o g e n b a l a n c e , which i s of s h o r t d u r a t i o n . These o b s e r v a t i o n s may be taken as i n d i c a t i n g t h a t c a r b o h y d r a t e does play a part  i n p r o t e i n metabolism which cannot be taken by f a t . 91  Munro, et a l  have suggested t h a t the mechanism through which  c a r b o h y d r a t e has an e f f e c t of p r o t e i n u t i l i z a t i o n may  be r e l a t e d to  the f a c t t h a t d i e t a r y c a r b o h y d r a t e , but not f a t , causes fall  i n plasma amino a c i d s and the d e p o s i t i o n  muscle.  The mechanism c a u s i n g  the s h i f t  has been a t t r i b u t e d to the a c t i o n of  attemporary  of amino a c i d s  of amino a c i d s 91 insulin.  i n the  i n t o muscle  44.  As a r e s u l t of the reduced plasma amino a c i d l e v e l , the supply  o f amino a c i d s t o o t h e r  t i s s u e s i s c u r t a i l e d and w i t h  two consequences: (a)  Urea p r o d u c t i o n action),  (b)  Tissues  by the l i v e r  i s reduced  (protein-sparing  and, other  than muscle haveaa dimished supply o f 91  amxno a c i d s f o r p r o t e i n syntheses.  '  Whether the i n s u l i n a c c e l e r a t e s the t r a n s p o r t of amino a c i d s across  the muscle c e l l membrane making the amino a c i d s a v a i l a b l e  to the p r o t e i n s y n t h e t i c mechanism w i t h i n the c e l l or whether the effect  i s on the p o l y p e p t i d e 101  clarified.  s y n t h e s i s w i t h i n the c e l l has not been  45.  I l l M a t e r i a l s and Methods PART A:  Feeding T r i a l  An experiment, e x t e n d i n g from November 3, 1966 1966 was  to December 7,  d e s i g n e d u s i n g a s i m p l e randomized b l o c k d e s i g n to study the  e f f e c t s of f e e d i n g r e g u l a r ranch r a t i o n s v a r y i n g on the l e v e l of p r o t e i n and energy d u r i n g  the n o n - c r i t i c a l p e r i o d of f e e d i n g .  The p r o t e i n and  energy c o n t e n t of the r a t i o n s were r e g u l a t e d by a l t e r i n g the p r o t e i n and energy c o n t e n t of the c e r e a l p o r t i o n . 1.  Materials a.  Animals - Two  hundred mature male and female mink were d i v i d e d  i n t o 5 groups w i t h 20 males and 20 females per group.  Due  to the  r e s t r i c t e d a v a i l a b i l i t y of any one c o l o u r phase of mink, a v a r i e t y of c o l o u r phased animals were used.  An attempt, t h e r e f o r e , was made to  d i s t r i b u t e the d i f f e r e n t c o l o u r phases e q u a l l y w i t h i n each group to e q u a l i z e any c o l o u r phase i n t e r a c t i o n between the groups. b.  Housing - A l l animals were i n d i v i d u a l l y housed  (17" x 15" x 15") equiped w i t h a wooden n e s t box a water cup.  i n w i r e cages  (8.5" x 7" x 7.5")  and  A l l cages were a d e q u a t e l y s h e l t e r e d a t the U.B.C. mink  unit. c.  Feed - Each of the 5 groups r e c e i v e d s i m i l a r r a t i o n s , ( T a b l e VI)  which v a r i e d o n l y i n the c e r e a l mix used. VIII) incorporated  The c e r e a l mixes  i n t o the r a t i o n s d i f f e r e d  energy, namely i n the form of f a t .  (Tables V I I and  i n l e v e l of p r o t e i n and  A l l r a t i o n s were mixed and f e d d a i l y  TABLE VI GENERAL COMPOSITION OF RATIONS  Amount  (c/lb)  Price  Constituent  (Per Cent)  C e r e a l Mix'''  25.8  Fish  25.8  3.5  25.8  4.5  17.5  11.0  4.1  10.0  1.0  19.5  Scraps  Chicken  Wastes  Horse Meat Liver Brewer's Yeast 2 V i t a m i n Premix S a l t Mix Water  Cost o f R a t i o n  Control 6.86  M-1A  M-1B  M-1C  M-1D  7.68  8.34  7.08  6.49  80 gms/lOOlbs o f r a t i o n - 120.0 20 gms/lOOlbs o f r a t i o n 151bs. o f ^ 0  (c/lb)  (as f e d )  3.1  added per 851bs. of above mixed ingredients.  Control 5.90  M-1A  M-1B  M-1C  M-1D  6.08  6.23  5.96  5.81  1.  See T a b l e s V I I and V I I I .  2.  Per gram : V i t a m i n A 550 I.U., V i t a m i n D 110 I.U., V i t a m i n E (d,alpha t o c o p e r y l a c e t a t e ) 0.51 I.U., C i t r u s B i o f l a v o n o i d compound 3.5 mg., M e t h i o n i n e 6.3 mg., C h o l i n e 6.3 mg., I n o s i t o l 2.1 mg., Thiamine HC1 0.1 mg., R i b o f l a v i n 0.3 mg., P y r i d o x i n e 0.03 mg., V i t a m i n B.^ 0.4 mg., N i a c i n 1.2 mg., d, p a n t o t h e n i c a c i d 0.1 mg., T r i c a l c i u m phosphate 48.0 mg., potassium b i phosphate 22.0 mg., Sodium c h l o r i d e 17.7 mg.  TABLE V I I THE COMPOSITION OF THE CONTROL CEREAL OF PART A  Amount (lbs) Constituent Corn Meal  350  Ground B a r l e y  200  Ground Wheat  335  Bran  100  Shorts  100  Soybean Meal (44%)  330  Fishmeal  (71%)  60  Meatmeal (50%)  285  V i t a Grass  40  Tomatoe Pomace  50  Brewer's Yeast  40  D i s t i l l e r s ' Dried Solubles  40  Stabilized Fat  55  Salt  10  V i t a m i n Premix  5  TABLE V I I I THE COMPOSITION OF EXPERIMENTAL CEREAL MIXES OF PART A  Constituents  M-1A (lbs)  Amounts M-1B M-1C (lbs) (lbs)  M-1D (lbs)  Ground Wheat  300  300  525  650  Ground Oats  300  300  525  650  Brewers' Y e a s t  60  60  40  40  Salt  20  20  20  20  H e r r i n g Meal  600  600  500  500  Soybean O i l Meal  500  500  100  100  40  40  40  180  250  5  5  Blood Meal  80  Meat Meal  100  Bone Meal  40  Stabalized Fat V i t a m i n Premix^  1.  5  5  Dry V i t a m i n A 1,000,000 I.U., V i t a m i n D 240,000 I.U., V i t a m i n E 28 gms., Thiamine m o n o n i t r a t e 2 gms., R i b o f l a v i n 7 gms., N i a c i n 12 gms., P y r i d o x i n e 5 gms., B i o t i n 0.1 gms., F o l i c A c i d 1.5 gms., Manfanese S u l f a t e 18.0 gms., D a c a l 6.0 l b s . , A l f a l f a l e a f m e a l 93.0 l b s .  49.  i n the a f t e r n o o n .  The raw meats were s t o r e d a t a temperature of  a p p r o x i m a t e l y -20°C.  Twenty-four hours p r i o r t o m i x i n g , the d a i l y  a l l o t m e n t s o f meat were removed from the f r e e z e r and a l l o w e d to thaw.  Water  was s u p p l i e d ad l i b i t u m .  2.  Methods a.  Animals - The day p r i o r t o the f e e d i n g of the e x p e r i m e n t a l  r a t i o n s , a l l mink were i n d i v i d u a l l y weighed recorded.  and t h e i r body weights  At the c o m p l e t i o n o f the experiment a l l mink were  sacrificed  by an i n t e r p e r i t o n e a l i n j e c t i o n of 1 ml. o f n i c o t i n e s u l f a t e (20% s o l u t i o n ) and weighed. b.  Feeds - The percentage d r y m a t t e r , p r o t e i n and e t h e r  extract  of the c e r e a l s and t o t a l r a t i o n s were determined u s i n g the o f f i c i a l 67 A.O.A.C. methods c. S t a t i s t i c a l A n a l y s i s - I n i t i a l body weight, f i n a l body weight, and body weight g a i n were a n a l y s e d u s i n g the A n a l y s i s o f V a r i a n c e and 132 Duncan's New M u l t i p l e Range T e s t  PART B 1. F e e d i n g T r i a l To g a i n a d d i t i o n a l i n f o r m a t i o n on the p r o t e i n / C a l o r i e  require-  ments o f mink, an experiment, e x t e n d i n g from J u l y 8, 1967 t o October 31, 1967 was designed (simple randomized b l o c k d e s i g n ) t o study the e f f e c t s of f e e d i n g r a t i o n s o f v a r y i n g p r o t e i n and energy l e v e l s t o mink k i t s  50.  d u r i n g the c r i t i c a l p e r i o d of f e e d i n g . content  Animals - Two  randomly d i s t r i b u t e d  energy  contents.  hundred and  eighty p a s t e l and  standard  i n t o 7 groups w i t h 40 k i t s per group.  k i t s were  Twenty males  twenty females were i n each group. ii.  Housing - A l l animals were housed i n cages as d e s c r i b e d i n P a r t A  of t h i s study.  Due  to the l a c k of space, however, one-half  animals on experiment had  to be housed two  per cage.  were removed from the cages on approximately iii.  Feed - The  i n composition The  energy  Materials i.  and  p r o t e i n and  of the t o t a l r a t i o n s were a g a i n r e g u l a t e d by u s i n g c e r e a l mixes  w i t h d i f f e r e n t p r o t e i n and a.  The  August 1,  the  n e s t boxes  1967.  c e r e a l mixes were i n c o r p o r a t e d i n t o r a t i o n s i d e n t i c a l  to the r a t i o n s g i v e n i n P a r t A  (Table VI)  c e r e a l mixes (Table IX) v a r i e d i n p r o t e i n and  the p r e v i o u s  The  of  experiment, however, i n t h i s study  of t h i s  energy content  the p r o t e i n and  d e n s i t y of the r a t i o n s were i n c r e a s e d to a l l o w f o r the i n c r e a s e demands of growth.  The  p r e p a r a t i o n and m i x i n g of the  r a t i o n s i s d e s c r i b e d i n P a r t A of t h i s b.  study. as i n calorie nutritive  experimental  study.  Methods Data c o l l e c t i o n , feed and  P a r t A of t h i s study. determination  One  s t a t i s t i c a l a n a l y s i s are d e s c r i b e d i n  method i n c l u d e d was  of the t o t a l r a t i o n s and  bomb c a l o r i m e t e r .  the gross  energy  c e r e a l mixes u s i n g a P a r r  oxygen  Thermethod i s d e s c r i b e d i n the P a r r t e c h n i c a l manual  51.  TABLE IX THE COMPOSITION OF EXPERIMENTAL CEREAL MIXES OF PART •B  M-2C lbs.  Amounts M-2D lbs.  M-2E lbs.  M-2F lbs.  DS-i lbs  Constituent  M-2A lbs.  M-2B lbs.  Ground Wheat  420  370  250  445  430  170  450  Ground  525  370  250  550  500  500  450  Brewers' Yeast  50  35  50  50  50  50  50  Salt  20  20  20  20  20  20  20  H e r r i n g Meal (71%)  300  725  800  500  540  465  550  Soybean O i l Meal (48.5%)  425  250  350  250  250  200  250  Meatmeal  90  90  140  140  100  360  90  Bonemeal  55  40  40  40  40  100  40  115  100  100  5  70  135  100  5  5  5  5  5  5  5  Oats  Stabalized Fat V i t a m i n Premix^  1.  Per Ton : V i t a m i n A 4,800,000 I.U., V i t a m i n D 240,00 I.U., V i t a m i n E 28.0 gm., Thiamine 3.2 gm., R i b o f l a v i n e 5.4 gm., P a n t o t h e n i e a c i d 22.4 gm., N i c o t i n e A c i d 27.5 gm., P y r i d o x i n e 3.2 gm., F o l i c a c i d 0.5  52.  2.  Digestibility  Trials  Two d i g e s t i b i l i t y  t r i a l s , each o f a 7 day d u r a t i o n , were  c a r r i e d out on the r a t i o n s used i n P a r t B o f Study I I . The t o t a l c o l l e c t i o n method was used w i t h and  the a d a p t a t i o n p e r i o d l a s t i n g 3 days  the c o l l e c t i o n p e r i o d c o n t i n u e d  f o r 4 days.  c o l l e c t i o n p e r i o d the f e c e s and u r i n e e x c r e t e d recorded.  The f e c e s was c o l l e c t e d on a w i r e  125 ml. f l a s k c o n t a i n i n g 2 ml. of 50% H S0.. 2 4 o  were pooled  Materials  i.  Animals - I n each t r i a l ,  cage.  and feed consumed were  s c r e e n and the u r i n e i n a The u r i n e and f e c e s  foranalysis,  a.  or Standard  Each day of the  7 male and 7 female p a s t e l ( T r i a l 1)  ( T r i a l 2) a d u l t mink were t r a n s f e r r e d to a d i g e s t i b i l i t y .--  T h i s was d e s i g n a t e d  as Day 1 o f the d i g e s t i b i l i t y  animals were weighed b e f o r e and a f t e r the 7 day a d a p t i o n  trial.  The  and c o l l e c t i o n  period. ii.  D i g e s t i b i l i t y Cages - The cages used were c o n s t r u c t e d 125  s p e c i f i c a t i o n s as o u t l i n e d by S i n c l a i r and Evans digestibility  trial  the cages were t h o r o u g h l y  hot water and scrubbed w i t h a w i r e iii.  .  Before  t o the  each  cleaned w i t h d e t e r g e n t and  brush.  Feeds - The f r o z e n meats used i n the r a t i o n s were allowed t o  thaw f o r twenty-four hours p r i o r t o g r i n d i n g . the feed and a l s o t o prevent  To ensure u n i f o r m i t y o f  the mink from c a r r y i n g l a r g e p i e c e s of  feed away from the f e e d i n g a r e a , the meats were put through a g r i n d e r twice.  S u f f i c i e n t q u a n t i t i e s of the r a t i o n s were mixed f o r the e n t i r e  t r i a l on Day 1 and s t o r e d under r e f r i g e r a t i o n u n t i l f e d . Each r a t i o n was fed  t o one male and one female mink.  53.  b.  Methods A l l n i t r o g e n , e t h e r e x t r a c t and d r y matter  were done u s i n g the o f f i c i a l A.O.A.C. procedures'^.  determinations Gross energy o f the  feeds and f e c e s were determined by oxygen bomb calorimetry"'"^.  54. IV.  Results  and D i s c u s s i o n  The mean i n i t i a l ,  f i n a l , and body weight g a i n s  of the  animals used i n P a r t A o f Study I I a r e shown i n Table X. record For  of i n i t i a l  A complete  and f i n a l body weights i s g i v e n i n Appendix I I I .  the purpose of t h i s experiment the c r i t e r i o n f o r a maintenance  r a t i o n was d e f i n e d  as t h a t r a t i o n which, when f e d ad l i b i t u m , caused  n e i t h e r a s i g n i f i c a n t i n c r e a s e nor decrease i n i n i t i a l body weight. The  levels  Table  o f p r o t e i n and f a t o f the v a r i o u s  XI.  F o r purposes o f c l a r i t y ,  are. c l a s s i f i e d  as f o l l o w s :  protein/high  f a t , M-lC;  protein/high  f a t , M-lB.  rations are given i n  the f o u r experimental  a) low p r o t e i n / l o w  c) h i g h p r o t e i n / l o w  f a t , M-lD;  rations b) low  f a t , M-1A; and d) h i g h  The r a t i o n c l a s s i f i e d  as the c o n t r o l was  the U.B.C. ranch r a t i o n (1960 t o 1966) and was employed i n t h i s experiment f o r the purpose of comparison.  The  r a t i o n M-lC (low p r o t e i n / h i g h f a t and r a t i o n M-lB (high  p r o t e i n / h i g h f a t ) supported g r e a t e r weight g a i n s  Table X.  Ration  to give  significantly  Mean I n i t i a l , F i n a l and Body Weight Gains of Study I I , P a r t A Initial Body Wt. (gm.)  Final Body Wt. (gm.)  Body Wt. Gain (gm.)  Control  1203  1218  15  M-1A  1233  1311  79  M-lB  1263  1339  76  M-lC  1260  1338  79  M-lD  1220  1218  -2  55. TABLE X I . . Proximate  Cereal  Percent Dry Matter  Ration  A n a l y s i s o f Rations P a r t A.  Mix  f o r Study I I ,  Total  Percent Ether Percent Extract Protein B a sis) (D.M.  Percent Dry Matter  Ration  Percent E ther Percent Extract Protein (D.M. B a s i s )  Control  88.56  27.02  10.19  30.75  43.03  23.76  M-1A  84.13  40.96  7.42  34.62  44.15  24.20  M-1B  88.46  37.27  19.87  35.27  44.75  28.87  M-1C  84.02  26.05  19.05  33.47  40.57  27.17  M-1D  83.68  26.71  11.08  32.00  36.64  21.25  All  v a l u e s a r e averages  greater  of t r i p l i c a t e  (P <0.05) f i n a l body weights  determinations.  over the c o n t r o l and M-lD r a t i o n s ,  of which r a t i o n M-lD i s low i n p r o t e i n and f a t .  R a t i o n M-1A  p r o t e i n / l o w f a t ) d i d n o t produce f i n a l body weights nificantly  (high  which d i f f e r e d  sig-  (P <0.05) from e i t h e r r a t i o n s M-lD and c o n t r o l or M-1B and  M-1C.  The M-lD r a t i o n appears  t o be the most s a t i s f a c t o r y  to m a i n t a i n mink near a c o n s t a n t body weight. r a t i o n s c o n t a i n i n g approximately on a d r y matter  of mink or over the maintenance p e r i o d .  because the animals  T h i s would suggest  that  36 p e r c e n t p r o t e i n and 21 p e r c e n t f a t ,  basis, are s a t i s f a c t o r y  f a t content would subsequently  ration  f o r the l a t e growth  requirements  R a t i o n s h i g h e r i n p r o t e i n or  be h i g h e r energy  r a t i o n s and as a r e s u l t ,  a r e i n t h e l a t t e r stages o f growth, would d e p o s i t  e x c e s s i v e amounts of body f a t , a c o n d i t i o n u n d e s i r a b l e f o r e i t h e r proper p e l t p r o c e s s i n g or b r e e d i n g  In conducted  purposes.  P a r t B o f t h i s study a more d e t a i l e d i n v e s t i g a t i o n was  t o determine  the p r o t e i n and energy  d u r i n g t h e i r growing p e r i o d . are shown i n T a b l e X I I . on a dry matter  o f mink  a n a l y s e s o f the 7 r a t i o n s  The percent p r o t e i n and f a t ( e t h e r e x t r a c t )  b a s i s ranged  percent, r e s p e c t i v e l y .  The proximate  requirements  from 38.9 t o 54.3 p e r c e n t and 18.3 t o 22.8  The mean i n i t i a l  body weight,  final  body  weight and body weight g a i n s a r e g i v e n i n Table X I I I and c a l c u l a t e d from the v a l u e s i n Appendix IV. found  t h a t the i n i t i a l  Table X I I .  body weights  Ration  o f the mink used  i n this  study  Proximate A n a l y s i s o f R a t i o n s f o r Study I I , Part B 3  Cereal  Percent Dry Matter  From an a n a l y s i s of v a r i a n c e i t was  Mix  Total  Percent Ether Percent Protein Extract (D.M. B a s i s )  Percent Dry Matter  Ration  Percent Percent Ether^ Protein Extracl (D.M B a s i s )  Control  92.18  36.70  12.53  37.10  42.42  20.55  M-2A  92.16  32.11  9.99  39.93  38.95  18.27  M-2B  91.64  42.09  10.78  35.43  46.01  18.38  M-2C  90.85  47.46  13.26  34.87  54.31  18.94  M-2D  92.31  40.49  10.50  37.92  48.10  20.38  M-2E  91.65  37.51  9.53  37.80  45.01  19.46  M-2F  90.23  39.18  14.27  34.99  47.87  22.78  Each v a l u e i s the mean v a l u e from t r i p l i c a t e  determinations  57. Table X I I I .  Mean I n i t i a l , F i n a l , and Body Weight Gains of Study I I , P a r t B  Initial Body Wt. (gm. )  Ration  Final Body Wt. (gm. )  a  Body Wt. Gain (gm. )  Control  553.6  1160  606.7  M-2A  569.0  1052  482.7  M-2B  582.9  1124  541.9  M-2C  583.4  1098  514.2  M-2D  667.3  1098  430.8  M-2E  627.7  1016  389.1  M-2F  660.3  1119  459.3  Significantly  different  (P<.0.01)  were s i g n i f i c a n t l y d i f f e r e n t  (P<0.01).  The f i n a l and i n i t i a l  body  weights were t h e r e f o r e t e s t e d u s i n g the A n a l y s i s of C o v a r i a n c e (62). A significant  difference  by t h i s a n a l y s i s . weight was  M-2D  and M-2F  The  i n f i n a l body weight was  ration  resulting  i n s i g n i f i c a n t l y greater  f i n a l body weights produced by r a t i o n s M-2B,  were not s i g n i f i c a n t  (P< 0.05).  R a t i o n M-2A  produce a f i n a l body weight s i g n i f i c a n t l y d i f f e r e n t t h a t produced by r a t i o n M-2E, nificantly different by the other  obtained  The r a t i o n which caused the g r e a t e s t g a i n s i n  the c o n t r o l  body w e i g h t s .  (P< 0.01)  rations.  but t h i s  M-2C,  d i d not  (P<< 0.05)  f i n a l body weight was  final  from sig-  (P<0.05) from the f i n a l body weights produced  The d e t a i l e d a n a l y s i s of the d i g e s t i b i l i t y  s t u d i e s and  n i t r o g e n r e t e n t i o n c a l c u l a t i o n s are g i v e n i n Appendices Summaries  of the d i g e s t i b i l i t y  trials  I t would be hazardous lications  i n the d i g e s t i b i l i t y  V and V I .  a r e g i v e n i n Tables XIV and XV.  t o suggest from the l i m i t e d reptrials  t h a t any one r a t i o n was  due t o a l a r g e r apparent d i g e s t i b i l i t y  coefficient.  superior  The d a t a i n  T a b l e s XIV and XV do show, however, d e f i n i t e ranges i n apparent d i g e s t i b l e dry matter, n i t r o g e n , e t h e r e x t r a c t and energy In both t r i a l s  the apparent dry matter d i g e s t i b i l i t y  averaged between 61.2 and 71.6%; nitrogen  d i g e s t i b l e energy  digestible  averaged between 83.0 and 91.4% and the apparent  (A.D.E.) v a r i e d between 69.6 and 77.9%, of the g r o s s  and t h a t f a t s a r e d i g e s t e d more e f f i c i e n t l y  The apparent d i g e s t i b i l i t y reported s l i g h t l y  apparent d i g e s t i b i l i t y  respectively.  f o r f a t and p r o t e i n have been  F o r example, Roberts and K i r k coefficients  74.82%, r e s p e c t i v e l y . crude p r o t e i n  coefficients  than p r o t e i n (81  h i g h e r i n e a r l y growth mink r a t i o n s than those  o b t a i n e d i n t h i s study.  fat  the p e r c e n t apparent  I t has been r e p e a t e d l y shown t h a t mink u t i l i z e f a t  efficiently  and  of the 7 r a t i o n s  (A.D.N.) averaged between 70.1 and 78.1, the apparent e t h e r  extract d i g e s t i b i l i t y  energy.  coefficients.  (106) o b t a i n e d  f o r f a t and p r o t e i n of 97.96 and  Bernard,, e t a l . (12) o b t a i n e d v a l u e s f o r f a t  ( c h i e f source raw horse meat) of 93.0 and 87.0%,,  Leoschke  (81) c o r r e c t e d  the apparent d i g e s t i b i l i t y  of  f o r the m e t a b o l i c f a t p r e s e n t i n the faeces of an e a r l y growth  r a t i o n and o b t a i n e d a t r u e d i g e s t i b i l i t y t a l l o w ) o f 91.9%.  coefficient  f o r f a t (beef  The average apparent d i g e s t i b l e dry m a t t e r i n t h i s  59. T a b l e XIV.  Summary of D i g e s t i b i l i t y  Trial I  3  Percent Nitrogen Digestibility Male Female  Percent Ether Extract Digestibility Male Female  Percent Digestible Energy Male Female  C o n t r o l 66.08 68.20 (mean)' (69.76)  72. 00  82.67 (78.1)  77. 21  86.80 (86.81)  67. 82  81. 01 (76. 96)  M-2A (mean )  56. 14  72.45 (61.26)  69. 12  78.42 (70.1)  82. 61  93.73 (86.51)  65. 17  77. 75 (69. 6)  M-2B (mean)  68. 24  73.11 (71.6)  73. 93  79.68 (76.91)  88. 26  92.40 (90.10)  73. 96  82. 61 (77. 91)  M-2C (mean)  66. 21  67.44 (68.7)  79. 55  80.41 (76.81)  72. 50  94.59 (87.2)  74. 86  79. 01 (76. 6)  M-2D (mean)  68. 10  66.82 (69.81)  72. 45  84.86 (77.10)  61. 73  92.39 (83.0)  73. 61  83. 49 (77. 91)  M-2E (mean)  63. 90  70.78 (69.31)  64. 95  81.44 (72.6)  90. 20  88.49 (91.4)  65. 07  81. 68 (76. 04)  M-2F (mean)  69. 66  65.08 (62.71)  81. 76  76.47 (71.6)  71. 13  90.09 (85.5)  82. 55  78. 15 (75. 01)  Percent D.M. Digestibility Male Female  Ration  3  All  v a l u e s a r e apparent- d i g e s t i b i l i t i e s  Mean o f d i g e s t i b i l i t y  trial  I and I I  60.  From the d a t a p r e s e n t e d i n Tables X I I I and XVI, i t i s e v i d e n t t h a t as the A.D.E. i s r a i s e d  from 353.4 Kcal/100 gm. d r y  matter t o 426.0 K c a l / l o o gm. and as the A.D.N, l e v e l reaches 4.980 gm./100 gm. d r y m a t t e r the e a r l y growth o f both male and female mink i s improved.  Maximum performance was o b t a i n e d i n the mink when the  r a t i o n c o n t a i n e d 426.0 K c a l A.D.E./100 gm. dry m a t t e r and 4.980 gm. A.D.N./100 gm. d r y matter. reported s i m i l a r r e s u l t s t h a t d i e t s h i g h i n energy  Sinclair,  e t . a l . (126), i n 1962,  t o those o b t a i n e d i n t h i s  (5.23 K c a l o f g r o s s energy/gm;  A.D.E./gm.) were s u p e r i o r t o low energy  Control  4.18 K c a l of  e a r l y growth and enhancing  i n male and female mink.  Table XVI.  Ration  He found  (4.57 K c a l o f g r o s s energy/gm;  3.43 K c a l of A.D.E./gm.) d i e t s i n promoting f i n a l body weights  study.  The Apparent D i g e s t i b l e Energy : Apparent D i g e s t i b l e N i t r o g e n R a t i o f o r the E x p e r i m e n t a l R a t i o n s as Determined from D i g e s t i b i l i t y T r i a l I I  Gross Energy (Kcal/100 gm. feed)  Apparent Digestible Energy (Kcal/100 gm. feed)  Gross Total CalrProtein Nitrogen Ratio (gm./100 feed) (Kcal/gm.)  Apparent Digestible Nitrogen (gtn./LOO gm. feed)  A.D.E. : A.D.N. Ratio (Kcal/gm.)  522.2  353.4  5.520  15.14  3.455  102.29  525.4  407.2  6.914  12.16  5.327  76.44  552.1  420.6  7.061  12.51  5.199  80.90  526.0  406.1  6.221  13.53  4.699  86.42  527.1  414.9  6.955  12.13  5.002  82.95  520.0  362.5  7.004  11.88  4.482  80.88  535.9  426.0  6.322  13.56  4.980  85.54  61. A l s o , d i e t s o f h i g h crude p r o t e i n  (4.912 gm. g r o s s N/100 gm;  A.D.N./100 gm.) were s u p e r i o r t o those o f low crude p r o t e i n (3.760 gm. g r o s s N/100 gm;  e t . a l . (126) a l s o found  t i b l e n i t r o g e n was c u r v i - l i n e a r r e l a t e d appeared The  t o be maximal when the d i e t  results  content  2.673 gm. A.D.N./100 gm.) i n a b i l i t y t o  promote e a r l y growth and t o enhance f i n a l body weights  Sinclair,  3.952 gm.  o f both  sexes.  t h a t the p e r c e n t d i g e s -  t o the A.D.E. : A.D.N. r a t i o and  c o n t a i n e d 140 K c a l of A.D.E./gm. A.D.N.  o b t a i n e d i n t h i s study do n o t show a c u r v i l i n e a r  s h i p between p e r c e n t A.D.N. and the A.D.E. : A.D.N. r a t i o however, a maximum response was o b t a i n e d when the d i e t  relation-  (Fig. I I ) ;  contained  85.54 K c a l of A.D.E./gm. A.D.N. (13.6 K c a l g r o s s energy/gm. g r o s s  Although results  protein).  a narrow range of A.D.E. : A.D.N. r a t i o s were s t u d i e d ,  from the n i t r o g e n b a l a n c e s t u d i e s (Appendices  VI) i n d i c a t e that  the A.D.E. content o f the d i e t had a d e f i n i t e p r o t e i n - s p a r i n g e f f e c t Table XVII). Table  Maximum n i t r o g e n r e t e n t i o n was obtained when  XVII.  The p r o t e i n - s p a r i n g e f f e c t o f i n c r e a s i n g apparent d i g e s t i b l e energy* 1  Ration  "  gm.)  Average N i t r o g e n R e t a i n e d (g.m.)  426.0  27.7  C  420.6  11.7  E  414.9  13.4  B  407.2  14.0  D  406.1  13.9  F  362.5  10.0  A  353.4  Control  a  A.D.E. (Kcal/100  9.3  A l l v a l u e s a r e averages o f 2 r e p l i c a t i o n s as determined from d i g e s t i b i l i t y , t r i a l I I .  62.  r a t i o n c o n t a i n e d 426.0 K c a l of A.D.E./100 gm. o f dry feed and as the energy  level  of the r a t i o n was reduced more p r o t e i n was m e t a b o l i z e d  as a source o f energy, retained.  i n d i c a t e d by the reduced amount o f n i t r o g e n  64.  Conclusions 1.  R a t i o n s c o n t a i n i n g 35 to 40% crude p r o t e i n  and  20 to 25% f a t , on a dry matter b a s i s , are s a t i s f a c t o r y f o r the l a t e growth and maintenance requirements  of  mink. 2.  The mean apparent d i g e s t i b i l i t y  c o e f f i c i e n t s obtained  i n these s t u d i e s f o r dry matter, p r o t e i n , f a t , and energy 3.  are 66.38, 74.05, 87.20, and  I t i s suggested t h a t growing male k i t s of age)  are capable of u t i l i z i n g  than female k i t s 4.  (from 6-28  weeks  feeds more e f f i c i e n t l y  of the same age.  As the A.D.E. i s r a i s e d from 353.4 Kcal/100 matter  to 426.0 Kcal/100  reaches 4.980 gm./lOO gm.  gm.  gm.  dry  and as the A.D.N. l e v e l  dry matter the e a r l y growth  of both male and female mink i s 5.  73.74%, r e s p e c t i v e l y .  A maximum e a r l y growth r a t e was  improved. o b t a i n e d i n k i t s when  the r a t i o n c o n t a i n e d 85.54 K c a l of A.D.E./gm. A.D.N. (13.6 K c a l gross energy/gm. gross p r o t e i n ) . 6.  I n c r e a s i n g the A.D.E. content of the d i e t s had a d e f i n i t e protein-sparing effect.  Maximum n i t r o g e n r e t e n t i o n  o b t a i n e d when the r a t i o n c o n t a i n e d 426.0 K c a l of A.D.E./100 gm.  of dry f e e d .  was  65.  VI.  References  cited.  1.  A d l e r , J.H. 1962. A n t i - o e s t r o g e n i c a c t i v i t y i n a l f a l f a . Vet. Rec. 74:1148-1150.  2.  A d l e r , J.H. 1965. A n t i - o e s t r o g e n i c a c t i v i t y i n f a h l i c l o v e r , hay and oat hay. A c t a endocr., Copenh. 49:90-96.  3.  A l d e n , R.H. 1942a. Aspects of the e g g - o v a r y - o v i d u c t r e l a t i o n s h i p i n the a l b i n o r a t : I . Egg passage and development f o l l o w i n g ovariectomy. J . Exp. Z o o l . 90:159-170.  4.  A l d e n , R.H. 1942b. A s p e c t s of the egg-ovary-oviduct r e l a t i o n s h i p i n the a l b i n o r a t : I I . Egg development w i t h i n the o v i d u c t . J . Exp. Z o o l . 90:171-182.  5.  A l d e n , R.H. 1942c. The o v i d u c t and egg t r a n s p o r t i n the a l b i n o r a t . Anat. Rec. 84:137-161.  6.  A l l e n , M.R. and W.D. K i t t s . 1961. The e f f e c t of y e l l o w p i n e (pinus ponderosa l a v e s ) n e e d l e s on the r e p r o d u c t i v i t y of the l a b o r a t o r y female mouse. Can. J . Anim. S c i . 41:1-8.  7.  A l l e n , M.R., E.V. Evans, and I.R. S i b b a l d . 1964. Energy: p r o t e i n r a l a t i o n s h i p s i n the d i e t s of growing mink. Can. J . P h y s i o l . Pharmacol. 42:733-744.  8.  Astwood, E.B. 1938. A s i x - h o u r assay f o r the q u a n t i t a t i v e d e t e r m i n a t i o n of e s t r o g e n . E n d o c r i n o l o g y . 23:25-31.  9.  Banik, U.K. and G. P i n c u s . 1962. E f f e c t of s t e r o i d a l a n t i p r o g e s t i n s on i m p l a n t a t i o n of f e r t i l i z e d eggs of r a t s and mice. P r o c . Soc. Exp. B i o l and Med. 111:595-602.  10.  Banik, U.K. and G. P i n c u s . 1964. Estrogens and t r a n s p o r t of ova i n the r a t . P r o c . Soc. Exp. B i o l , and Med. 116:1032-1034.  11.  B e n n e t t s , H.W., C.J. Underwood, and F.L. S h i e r . 1946. A s p e c i f i c b r e e d i n g problem of sheep on s u b t e r r a n e a n c l o v e r p a s t u r e s i n Western A u s t r a l i a . Aust. V e t . J . 22:2-12.  12.  Bernard, R., S.E. Smith, and L.A. Maynard. 1942. D i g e s t i o n of c e r e a l s by minks and goxes w i t h s p e c i a l r e f e r e n c e t o s t a r c h and crude f i b e r . C o r n e l l V e t . 32:29-36.  66.  13.  B e y l e r , A.L. and G.O. P o t t s . 1957. The e f f e c t s of e t h i n y l a n d r o s t e n e d i o l 3 - c y c l o h e x y l p r o p i o n a t e on the r e p r o d u c t i v e t r a c t of male and female r a t s . Endocrinology. 60:519-531.  14.  B i c k o f f , E.M., A.N. Booth, R.L. Lyman, A.L. L i v i n g s t o n , C.R. Thompson, and F. DeEds. 1957. Coumestrol, a new e s t r o g e n i s o l a t e d from f o r a g e c r o p s . Science. 126:969-970.  15.  B i c k o f f , E.M., A.L. L i v i n g s t o n , A.N. Booth, A.P. Hendrickson, and G.O. K o h l e r . 1960. E s t r o g e n i c a c t i v i t y i n dehydrated and suncured f o r a g e s . J . Animal S c i . 19:189-197.  16.  B l a c k , D.L. and S.A. A s d e l l . 1968. T r a n s p o r t through oviduct. Amer. J . P h y s i o l . 192:63-68.  17.  B l a c k , D.L. and S.A. A s d e l l . 1959. Mechanism c o n t r o l l i n g e n t r y of ova i n t o r a b b i t u t e r u s . Amer. J . P h y s i o l . 197:1275-1278..  18.  Bosshardt, D.K., W. P a u l , K. O'Doherty, and R.H. Barnes. 1946. The i n f l u e n c e of c a l o r i c i n t a k e on the growth u t i l i z a t i o n of d i e t a r y p r o t e i n . J . N u t r i . 32:641-651.  19.  Bosshardt, D.K., W.J% P a u l , K. O'Doherty, and R.H. Barnes. 1948. C a l o r i c r e s t r i c t i o n and p r o t e i n n e t a b o l i s m i n the growing mouse. J . N u t r i t i o n . 36: 773-783.  20.  B u r d i c k , H.O., B.B. Emerson, and R. Whitney. 1940. Effects of t e s t o s t e r o n e p r o p i o n a t e on pregnancy and on passage of ova through the o v i d u c t s of mice. Endocrinology. 26:1081-1086.  21.  B u r d i c k , H.O. and G. P i n c u s . 1934. The e f f e c t of o e s t r i n i n j e c t i o n s upon the d e v e l o p i n g ova of mice and r a b b i t s . Am. J . P h y s i o l . 111:201-208.  22.  B u r d i c k , H.O. and H. Vedder. 1941. The e f f e c t of s t i l b e s t e r o l i n e a r l y pregnancy. Endocrinology. 28:629-632.  23.  B u r d i c k , H.O., R. Whitney. 1937. A c c e l e r a t i o n of the r a t e of passage of f e r t i l i z e d ova through the F a l l o p i a n tubes of mice by massive i n j e c t i o n s of an e s t r o g e n i c substance. Endocrinology. 21:637-643.  24.  B u r d i c k , H.O. and R. Whitney. 1938. Fate of ova a c c e l e r a t e d i n t h e i r r a t e of passage through the F a l l o p i a n tubes of mice by massive i n j e c t i o n s of progynon-B. E n d o c r i n o l o g y . 22:631-638.  rabbit  67.  25.  B u r d i c k , H.O., R. Whitney, and G. P i n c u s . 1937. The f a t e of mouse ova t u b e - l o c k e d by i n j e c t i o n s o f o e s t r o g e n i c substances. Anat. Rec. 67:513-519.  26.  Calloway, D.H. and H. S p e c t o r . 1954. N i t r o g e n b a l a n c e as r e l a t e d to c a l o r i c and p r o t e i n i n t a k e i n a c t i v e young men. Amer. J . C l i n . Nutr. 2:405-411.  27.  Calloway, D.H. and H. S p e c t o r . 1955 a. Nitrogen u t i l i z a t i o n during c a l o r i c r e s t r i c t i o n . I . The e f f e c t of d i e t a r y f a t content. J. Nutrition. 56:533-544.  28.  Calloway, D.H. and H. S p e c t o r . 1955b. N i t r o g e n u t i l i z a t i o n during c a l o r i c r e s t r i c t i o n . I I . The e f f e c t of v a r i a t i o n in nitrogen intake. J . Nutrition. 56: 545-554.  29.  C a r l s e n , R.A., G.H. Z e i l m a k e r , and M.C. Shelesnyak. 1961. T e r m i n a t i o n of e a r l y ( p r e - n i d a t i o n ) pregnancy i n the mouse by s i n g l e i n j e c t i o n of e r g o c o r n i n e methanesulphonate. J . Reprod. F e r t i l . 2:369-373.  30.  Chang, M.C, 1964. E f f e c t s of c e r t a i n a n t i f e r t i l i t y agents on the development of r a b b i t ova. F e r t . S t e r i l . 15:97-106.  31.  Chang, M.C. and R. Yanagimachi. 1965. E f f e c t of estrogens and o t h e r compounds as o r a l a n t i f e r t i l i t y agents on the development of r a b b i t ova and hamster embryos. F e r t . S t e r i l . 16:281-291.  32.  Cochrane, R.L. and R.M. S h a c k e l f o r d . 1962. E f f e c t s of exogenous o e s t r o g e n a l o n e and i n combination w i t h progesterone on pregnancy i n the i n t a c t mink. J . E n d o c r i n . 25:101-106.  33.  Cook, H. and W.D. K i t t s . 19,64. Anti-oestrogenic a c t i v i t y i n y e l l o w p i n e needles (Pinus Ponderosa) . A c t a . Endocr. 45:33-39.  34.  C o u r r i e r , R. 1950. I n t e r a c t i o n s between e s t r o g e n i c and p r o g e s t e r o n e . V i t a m i n s and Hormones. 8:179-214.  35.  C o u r r i e r , R. and A. J o s t . 1944. A c t i o n du p r o p i o n a t e de t e s t o s t e r o n e sur l a g e s t a t i o n chez l a l a p i n e . C.R. Soc. B i o l . Paris. 138:285.  36.  D'Amour, F.E., M.C. D'Amour, and R.G. Gustavson. 1933. E f f e c t s of e s t r i n and o t h e r hormones upon pregnancy. J . Pharmacol. 49:146-161.  68.  37.  D'Amour, F.E. and R.G. Gustavson. 1934. A h i s t o l o g i c a l study of t h e a c t i o n of e s t r i n i n t e r m i n a t i n g pregnancy. J . Pharmacol, and Exper. Therap. 51:353-359.  38.  D a n i e l , J r . , J.C. 1967. S t u d i e s on growth of t h e mink b l a s t o c y s t . J . Embryol. exp. Morph. 17:293-302.  39.  D a v i s , B.K. 1963. T e r m i n a t i o n o f pregnancy i n the r a t w i t h norethynodrel. Nature. 197:308-309.  40.  Deanesly, R. 1963. F u r t h e r o b s e r v a t i o n s on t h e e f f e c t s of o e s t r a d i o l on t u b a l eggs and i m p l a n t a t i o n i n t h e g u i n e a - p i g . J . Reprod. F e r t i l . 5:49-57.  41.  De Feo, V . J . 1963. Temporal aspects o f u t e r i n e s e n s i t i v i t y i i n the pseudopregnant or pregnant r a t . E n d o c r i n o l o g y . 72:305-316.  42.  Dodds, E.C., L. Goldberg, W. Lawson, and R. Robinson. 1938. O e s t r o g e n i c a c t i v i t y o f c e r t a i n s y n t h e t i c compounds. Nature. 141:247-248.  43.  Dodds, E.C., W. Lawson, and R.L. Noble. 1938. B i o l o g i c a l e f f e c t s of t h e s y n t h e t i c o e s t r o g e n i c substance 4:4'-dihydroxy- :£ 3 -diethylstilbene. Lancet. 1:1389-1391.  44.  D r e i s b a c h , R.H. 1959. The e f f e c t s of s t e r o i d sex hormones on pregnant r a t s . J . Endocrin. 18:271-277.  45.  Duncan, G.W. and A.D. Forbes. 1965. B l a s t o c y s t s u r v i v a l and n i d a t i o n i n r a t s t r e a t e d w i t h oestrogen a n t a g o n i s t s . J . Reprod. F e r t i l . 10:161-167.  46.  Edgar, D.G. and S.A. A s d e l l . 1960. The v a l v e - l i k e a c t i o n of the u t e r o - t u b a l j u n c t i o n o f the ewe. J . Endocrin. 21:315-320.  47.  Edgren, R.A. and S h i p l e y , G.C. 1961. A q u a n t i t a t i v e study of the t e r m i n a t i o n of pregnancy i n r a t s w i t h e s t r o n e . F e r t i l . and Steril. 12:178-181.  48.  Emmens, C.W. 1962. A c t i o n of oestrogens and a n t i - o e s t r o g e n s on e a r l y pregnancy i n t h e r a b b i t . J . Reprod. F e r t . 3:246-249.  49.  Emmens, C.W. 1965a. The o e s t r o g e n i c and a n t i f e r t i l i t y a c t i v i t i e s of d i e t h y l s t i l b e s t e r o l and d i m e t h y l s t i l b e s t e r o l i n r o d e n t s . A c t a endocr., Copenh. 49:83-89.  50.  Emmens, C.W. 1965b. O e s t r o g e n i c , a n t i = o e s t r o g e n i c and a n t i f e r t i l i t y a c t i v i t i e s o f v a r i o u s compounds. J . Reprod. F e r t i l . 9:277-283.  69.  51.  Emmens, C.W., R.I. Cox, and L. M a r t i n . 1962. Recent Prog. Horm. Res. 18:415-466.  Antiestrogens.  52.  Emmens, C.W., R.I. Cox, and L. M a r t i n . 1964. The o e s t r o g e n i c and a n t i - o e s t r o g e n i c a c t i v i t y o f compounds r e l a t e d t o diethylstilbesterol. A c t a endocr. Suppl. 90:61-69.  53.  Emmens, C.W.and C.A. F i n n . 1962. L o c a l and p a r e n t e r a l a c t i o n of o e s t r o g e n and a n t i - o e s t r o g e n s on e a r l y pregnancy i n the r a t and mouse. J . Reprod. F e r t i l . 3:239-245.  54.  Enders, R.K. and A.C. Enders. 1963. Morphology of t h e female r e p r o d u c t i v e t r a c t d u r i n g delayed i m p l a n t a t i o n i n t h e mink. In Delayed I m p l a n t a t i o n . A.C. Enders ( e d . ) . U n i v e r s i t y o f Chicago P r e s s , pp. 129-139.  55.  F i n n , C.A. and C.W. Emmens. 1961. The e f f e c t o f d i m e t h y l s t i l b e s t e r o l and o e s t r a d i o l on deciduoma f o r m a t i o n i n t h e r a t . J . Reprod. F e r ' t i l . 2:528-529.  56.  Fowler, E.R. and R.G. Edwards. 1960. E f f e c t s of p r o g e s t e r o n e and oestrogen on pregnancy and embryonic m o r t a l i t y i n a d u l t mice f o l l o w i n g s u p e r o v u l a t i o n treatment. J . Endocrin. 20: 1-8.  57.  Greenwald, G.S 1957. I n t e r r u p t i o n o f pregnancy i n t h e r a b b i t by the a d m i n i s t r a t i o n o f e s t r o g e n . J . exp. Z o o l . 135:461-470.  58.  Greenwald, G.S. 1959. The comparative e f f e c t i v e n e s s of estrogens i n i n t e r r u p t i n g pregnancy i n the r a b b i t . F e r t i l . and S t e r i l . 10:155-161.  59.  Greenwald, G.S. 1961a. A study of t h e t r a n s p o r t o f ova through t h e rabbit oviduct. F e r t i l . and S t e r i l . 12:80-95.  60.  Greenwald, G.S. 1961b. The a n t i - f e r t i l i t y e f f e c t s i n pregnant r a t s of a s i n g l e i n j e c t i o n o f e s t r a d i o l c y c l o p e n t y l p r o p i o n a t e . E n d o c r i n o l o g y . 69:1068-1073.  61.  Greenwald, G.S. 1963. I n t e r r u p t i o n of e a r l y pregnancy i n t h e r a b b i t by s i n g l e i n j e c t i o n of o e s t r a d i o l c y c l o p e n t y l p r o p i o n a t e . J. Endocrin. 26:133-138.  62.  Guenther, W.C. 1964. A n a l y s i s of V a r i a n c e . Englewood C l i f f s , N.J. pp. 143-166.  63.  Hafez, E.S.E. 1962. Endocrine c o n t r o l of r e c e p t i o n , t r a n s p o r t , development and l o s s of r a b b i t ova. J . Reprod. F e r t i l . 3:14-25.  Prentice-Hall.  70.  64.  Harper, M.J.K. 1964. The e f f e c t s of constant doses of o e s t r o g e n and p r o g e s t e r o n e on the t r a n s p o r t of a r t i f i c i a l eggs through the r e p r o d u c t i v e t r a c t of o v a r i e c t o m i z e d r a b b i t s . J. Endocrin. 30:1-19.  65.  Hartman, C G . 1939. O v u l a t i o n , f e r t i l i z a t i o n and the t r a n s p o r t and v i a b i l i t y of eggs and spermatozoa. In Sex and I n t e r n a l S e c r e t i o n s , 2nd ed. Ed. E. A l l e n .  66.  H e c k e l , G.P. and W.M. Allen. 1939. Maintenance of the corpus luteum and i n h i b i t i o n of p a r t u r i t i o n i n the r a b b i t by i n j e c t i o n of e s t r o g e n i c hormone. E n d o c r i n o l o g y . 24:137-148.  67.  H o r w i t z , W. (ed.) 1960. O f f i c i a l Methods of A n a l y s i s of the A s s o c i a t i o n of O f f i c i a l A g r i c u l t u r a l Chemists. 9th ed. A.O.A.C. Washington, D.C.  68.  Huggett, A. S t . G. and J . J . P r i t c h a r d . 1945. Experimental f o e t a l death: the s u r v i v i n g p l a c e n t a . J . Proc. Roy. Soc. Med. 38:261-266.  69.  Humphrey, K. 1967. The development of v i a b l e embryos a f t e r ovum t r a n s f e r s to long-term o v a r i e c t o m i z e d mice. S t e r o i d s . 9:53-56.  70.  Humphrey, K. and L. M a r t i n . 1968. The e f f e c t of oestrogens and a n t i o e s t r o g e n s on ovum t r a n s p o r t i n mice. J . Reprod. F e r t . 15: 191-197.  71.  Humphrey, K.W. 1968. O b s e r v a t i o n s on t r a n s p o r t of ova i n the o v i d u c t of the mouse. J . E n d o c r i n . 40:267-273.  72.  Jacks on, H. 1959. 135-172.  73.  Johnstone, T.H. and M.C Shelesnyak. 1958. Histamine-oestrogenp r o g e s t e r o n e complex a s s o c i a t e d w i t h the d e c i d u a l c e l l r e a c t i o n and w i t h ovum i m p l a n t a t i o n . J . E n d o c r i n . 17:xxi - x x i i .  74.  J o s t , A. 1945. A c t i o n du p r o p i o n a t e de t e s t o s t e r o n e sur l a g e s t a t i o n chez l a s o u r i s . C R . Soc. B i o l . P a r i s . 139:483-492.  75.  K e l l y , G.L. 1931. The e f f e c t of i n j e c t i o n s of female sex hormone ( o e s t r i n ) on c o n c e p t i o n and pregnancy i n the guinea p i g . Surg. Cynec. Obst. 52:713-722.  76.  K e t c h e l , M.M. and G. P i n c u s . 1964. In v i t r o exposure of r a b b i t to e s t r o g e n s . P r o c . Soc. exp. B i o l . Med. 115:419-421.  77.  K i t t s , W.D., E. S w i e r s t r a , V . C B r i n k , and A . J . Wood. 1959a. The e s t r o g e n - l i k e substances i n c e r t a i n legumes and g r a s s e s . I.' The q u a n t i t a t i v e d e t e r m i n a t i o n of such substances i n r e d c l o v e r and o a t s . Can. J . Anim. S c i . 39:6-13.  Antifertility  substances.  Pharmacol.  Rev.  11:  ova  71.  78.  K i t t s , W.D., E. S w i e r s t r a , V.C. B r i n k , and A . J . Wood. 1959b. The e s t r o g e n - l i k e substances i n c e r t a i n legumes and g r a s s e s . II. The e f f e c t of stage o f m a t u r i t y and frequency o f c u t t i n g on t h e e s t r o g e n i c a c t i v i t y of some f o r a g e s . Can. J . Anim. Sci. 39:158-163.  79.  K r a i c e r , P., and M.C. Shelesnyak. 1958. The i n d u c t i o n of deciduomata i n t h e pseudopregnant r a t by s y s t e m i c a d m i n i s t r a t i o n o f h i s t a m i n e and h i s t a m i n e r e l e a s e r s . J . E n d o c r i n . 17:324-328.  80.  Legg, S.P., D.H. Curnow, and S.A. Simpson. 1950. The s e a s o n a l and species d i s t r i b u t i o n of oestrogen i n B r i t i s h pasteur p l a n t s . Biochem. J . (London). 46:xix - xx.  81.  Leoschke, W.L. 1959. The d i g e s t i b i l i t y of animal f a t s and p r o t e i n s by mink. Am. J . V e t . Res. 20:1086-1089.  82.  L e v i n , L., P.A. Katzman, and E.A. Doisy - 1931. E f f e c t s of e s t r o g e n i c substances and the l u t e i n i z i n g f a c t o r on pregnancy i n the a l b i n o r a t . '-Endocrinology. 15:207-213.  83.  L i u z z o , J.A., J.G. Lee, A.B. Watts, E.A. F i e g e r , and A.F. Novak. 1960. S t i m u l a t i o n of c h i c k growth w i t h a l f a l f a c o n c e n t r a t e s . P o u l t r y Sci. 39:823-827.  84.  MacDonald, M.A. 1952. P i n e needle a b o r t i o n i n range beef J . Range Management. 5:150-155.  85.  M a r t i n , L. 1962. The e f f e c t s of h i s t a m i n e on the v a g i n a l e p i t h e l i u m of, the mouse. J . E n d o c r i n . 23:329-340.  86.  M a r t i n , L., R.I. Cox, and C.W. Emmens. 1963. Further studies on t h e e f f e c t s of oestrogens and a n t i - o e s t r o g e n s on e a r l y pregnancy i n mice. J . E n d o c r i n . 20:299-306.  87.  M a r t i n , L., C.W. Emmens, and R.I. Cox. 1960. The e f f e c t s o f oestrogens and a n t i - o e s t r o g e n s on e a r l y pregnancy i n mice. Endocrin. 20: 299-306.  cattle.  J.  88.  Meyer, R.K. and E.F. N u t t i n g . 1964. E f f e c t of combinations of p r o g e s t e r o n e and o e s t r o n e on t h e d e l a y o f n i d a t i o n , i m p l a n t a t i o n and f o e t a l s u r v i v a l i n o v a r i e c t o m i z e d r a t s . J . E n d o c r i n . 29:243-249.  89.  M o r r i s o n , A.B. and M. Narayana Rao. 1967. Some r e l a t i o n s h i p s between p r o t e i n s and c a l o r i e s . World Rev. Nutr. D i e t . 7:204-224.  90.  Munro, H.N. 1951.- Carbohydrate u t i l i z a t i o n and metabolism.  and f a t as f a c t o r s i n p r o t e i n P h y s i o l . Rev. 31:449-488.  72.  91.  Munro, H.N., J.G. B l a c k and W.S.T. Thompson. 1959. The mode of a c t i o n o f d i e t a r y c a r b o h y d r a t e on p r o t e i n metabolism. B r i t . J . Nutr. 13:475-485.  92.  Munro, H.N. and D.J. N a i s m i t h . 1953. The i n f l u e n c e of energy i n t a k e on p r o t e i n metabolism. Biochem. J . 54:191-197.  93.  Noyes, R.W., C.E. Adams, and A. Walton. 1959. The t r a n s p o r t of ova i n r e l a t i o n t o the dosage of o e s t r o g e n i n o v a r i ectomized r a b b i t s . J . E n d o c r i n . 18:108-117.  94.  N u t t i n g , E.F. and R.K. Meyer. 1964. E f f e c t of o e s t r o n e on the d e l a y of n i d a t i o n , i m p l a n t a t i o n and f o e t a l s u r v i v a l i n ovariectomized r a t s . J . Endocrin. 29:235-242.  95.  Ostrovsky., D. and W.D. K i t t s . 1962a. substances i n legumes and g r a s s e s . 42: 129-138.  96.  O s t r o v s k y , D. and W.D. K i t t s . 1962b. E s t r o g e n - l i k e substances i n legumes and g r a s s e s . Can. J . Biochem. P h y s i o l . , 40:159-164.  97.  O s t r o v s k y , D. and W.D. K i t t s . 1963. The e f f e c t o f e s t r o g e n i c p l a n t e x t r a c t s on the uterus of t h e l a b o r a t o r y r a t . Can. J . Anim. S c i . 43:106-112.  98.  P a r k e s , A.S. and C.W. B e l l e r b y . 1926. S t u d i e s on the i n t e r n a l s e c r e t i o n s o f t h e ovary. I I . • The e f f e c t s of i n j e c t i o n of the o e s t r u s p r o d u c i n g hormone d u r i n g pregnancy. J . Physiol. 62: 145-155.  99.  Parkes, A.S., E.C. Dodds, and R.L. Noble. 1938. I n t e r r u p t i o n of e a r l y pregnancy by means of o r a l l y a c t i v e o e s t r o g e n s . Brit. Med. J . 2: 557-559.  The e s t r o g e n - l i k e Gan. J . Anim. S c i . ,  100.  P a r r Instrument Co. 1960. Oxygen Bomb C a l o r i m e t r y and Combust i o n Methods. T e c h n i c a l Manual No. 130. P a r r Instrument Co. M o l i n e , I l l i n o i s .  101.  P i k e , R.L., and M.L. Brown. 1967. N u t r i t i o n : An I n t e g r a t e d Approach. John W i l e y and Sons, New York, pp 432-435.  102.  P i k e , R.L., H.B. Suder, and M.L. Ross. 1954. The i n f l u e n c e of p r o t e i n and energy i n t a k e s upon n i t r o g e n r e t e n t i o n i n t h e pregnant r a t . J . N u t r i . 52: 297-309.  103.  P i n c u s , G., U.K. Banik, and J . J . Jacques. 1964. F u r t h e r s t u d i e s on i m p l a n t a t i o n i n h i b i t o r s . Steroids. 4: 657-677.  104.  P i n c u s , G. and R.E. K i r s c h . 1936. The s t e r i l i t y i n r a b b i t s produced by i n j e c t i o n s of o e s t r o n e and r e l a t e d compounds. Amer. J . P h y s i o l . 115: 219-228.  73.  105.  R o b e r t s , W.K. 1950. What happens when p e l l e t - i n s e r t e d heads a r e fed? Amer. Fur Breeder. 23(4): 17-18.  chicken  106.  R o b e r t s , W.K. and R.J. K i r k . 1964. D i g e s t i b i l i t y and n i t r o g e n u t i l i z a t i o n of raw f i s h and d r y meals by mink. Amer. J . Vet. Res. 25: 1746-1750.  107.  R o s e n t h a l , H.L. 1952. The e f f e c t of d i e t a r y f a t and c a l o r i c r e s t r i c t i o n on p r o t e i n u t i l i z a t i o n . J . N u t r i t i o n , 48: 243-255.  108.  R o s e n t h a l , H.L. and J.B. A l l i s o n . 1951. Some e f f e c t s of c a l o r i c i n t a k e on n i t r o g e n b a l a n c e i n dogs. J . N u t r i t i o n , 44: 423-431.  109.  R o s e n t h a l , H.L. and J.B. A l l i s o n . 1956. E f f e c t s o f c a l o r i c i n t a k e on n i t r o g e n b a l a n c e and organ c o m p o s i t i o n o f a d u l t rats. J . A g r i . Food Chem. 4: 792-796.  110.  Runner, M.N. 1947. Development of mouse eggs i n the a n t e r i o r chamber o f the eye. Anat. Rec. 98: 1-13.  111.  Saunders, F . J . 1958. The e f f e c t s o f s e v e r a l s t e r o i d s on f e c u n d i t y i n female r a t s . Endocrinology, 63: 561-565.  112.  Saunders, F . J . 1964. Some notes on the mode of a c t i o n o f norethynodrel i n preventing f e r t i l i t y i n r a t s . Acta. Endocrinol. 46: 157-160.  113.  S c h o f i e l d , M.B. 1962. The e f f e c t of i n j e c t e d o e s t r o g e n on pregnancy i n the r a b b i t . J . E n d o c r i n . 25: 95-100.  114.  S e g a l , S.J. and W.O. N e l s o n . 1958. An o r a l l y a c t i v e compound with a n t i - f e r t i l i t y e f f e c t s i n r a t s . P r o c . Soc. Exp. B i o l . Med. 98: 431-436.  115.  S e g a l , S.J. and A. T y l e r . 1958. S t r u c t u r e - a c t i v i t y - r e l a t i o n s h i p s c o n c e r n i n g the i n h i b i t o r y a c t i v i t y o f s y n t h e t i c e s t r o g e n s and some t r i p h e n y l e t h a n o l d e r i v a t i v e s on d e v e l o p i n g eggs o f A r a b a c i a p u n c t u l a t a . B i o l . B u l l . , Woods Hole. 115: 364-365.  116.  Shelesnyak, M.C. 1952. I n h i b i t i o n of d e c i d u a l c e l l f o r m a t i o n i n the pseudopregnant r a t by h i s t a m i n e a n t a g o n i s t s . Amer. J. Physiol. 170: 522-527.  117.  Shelesnyak, M.C. 1955. D i s t u r b a n c e of hormone b a l a n c e i n the female r a t s by a s i n g l e i n j e c t i o n o f e r g o t o x i n e ethanesulphonate. Amer. J . P h y s i o l . 180: 47-49.  74.  118.  Shelesnyak, M.C. 1957. Some e x p e r i m e n t a l s t u d i e s on the mechanism of o v a - i m p l a n t a t i o n i n the r a t . Recent P r o g r . Hormone Res. 13: 269-322.  119.  Shelesnyak, M.C. 1959a. F a l l i n u t e r i n e h i s t a m i n e a s s o c i a t e d w i t h ovum i m p l a n t a t i o n i n pregnant r a t . P r o c . Soc. E x p t l . B i o l . Med. 100: 380-381.  120.  Shelesnyak, M.C. 1959b. H i s t a m i n e r e l e a s i n g a c t i v i t y of n a t u r a l e s t r o g e n s . P r o c . Soc. E x p t l . B i o l . Med. 100: 739-741.  121.  Shelesnyak, M.C. 1959c. H i s t a m i n e and the n i d a t i o n of the ovum. In Mem. Soc. E n d o c r i n . No. 6. I m p l a n t a t i o n of Ova. pp. 84-88.  122.  Shelesnyak, M.C. 1962. Decidualization: the d e c i d u a and deciduoma. P e r s p e c t . B i o l . Med. 5: 503-517.  123.  Shelesnyak, M.C, P.F. K r a i c e r , and G.H. Z e i l m a k e r . 1963. S t u d i e s on the mechanism of d e c i d u a l i z a t i o n . I. The o e s t r o g e n i c surge o f pseudopregnancy and p r o g r a v i d i t y and i t s r o l e i n the p r o c e s s of d e c i d u a l i z a t i o n . Acta Endocrin., Copenhagen. 42: 225-232.  124.  S i b b a l d , I.R., J.P. Bowland, A.R. Robblee, and R.T. Berg. 1957. Apparent d i g e s t i b l e energy and n i t r o g e n i n the food of the weanling r a t . J . N u t r i t i o n . 61: 71-85.  125.  S i n c l a i r , D.G. and E.V. f o r use w i t h mink.  126.  S i n c l a i r , D . C , E.V. Evans, and I.R. S i b b a l d . 1962. The i n f l u e n c e of apparent d i g e s t i b l e energy and apparent d i g e s t i b l e n i t r o g e n i n the d i e t on weight g a i n , f e e d consumption and n i t r o g e n r e t e n t i o n of growing mink. Can. J . Biochem. Physiol. 40: 1375-1389.  127.  Smith, D.M. 1968a. The e f f e c t of the time of e s t r o g e n i n j e c t i o n on i m p l a n t a t i o n i n o v a r i e c t o m i z e d pregnant mice. J . Endocrin. 4 1 ( 1 ) : 11-15.  128.  Smith, D.M. 1968b. The e f f e c t on i m p l a n t a t i o n of t r e a t i n g c u l t u r e d mouse b l a s t o c y s t s w i t h o e s t r o g e n i n v i t r o and the uptake of ( H) o e s t r a d i o l by b l a s t o c y s t s . J. Endocrin. 4 1 ( 1 ) : 17-29.  129.  Smith, D.M. and J.D. B i g g e r s . 1968. The o e s t r o g e n requirement f o r i m p l a n t a t i o n and the e f f e c t o f i t s dose on the i m p l a n t a t i o n response i n the mouse. J . E n d o c r i n . 4 1 ( 1 ) : 1-9.  Evans. 1962. A metabolism Can. J . Biochem. P h y s i o l .  the  cage designed 40: 1395-1399.  75.  130.  Smith, M.G. 1926. On t h e i n t e r r u p t i o n of pregnancy by t h e i n j e c t i o n o f o v a r i a n f o l l i c u l a r e x t r a c t . John Hopkins Hosp. 39: 203-214.  i n the r a t Bull.  131.  S p a z i a n i , E. and C M . Szego. 1958. The i n f l u e n c e of e s t r a d i o l and C o r t i s o l on u t e r i n e h i s t a m i n e o f the o v a r i e c t o m i z e d rat. E n d o c r i n o l o g y . 63: 669-678.  132.  S t e e l , R.G.D. and J.H. T o r r i e . 1960. P r i n c i p l e s and Procedures of S t a t i s t i c s . McGraw-Hill, New York. pp. 101.  133.  Stone, C M . and C.W. Emmens. 1964a. The a c t i o n o f o e s t r a d i o l and d i m e t h y l s t i l b e s t e r o l on e a r l y prenancy and deciduoma f o r m a t i o n i n t h e mouse. J . E n d o c r i n . 29: 137-145.  134.  Stone, C M . and C.W. Emmens. 1964b. The e f f e c t of oestrogens and a n t i - o e s t r o g e n s on deciduoma f o r m a t i o n i n t h e r a t . J . E n d o c r i n . 29: 147-157.  135.  T h i e r s c h , J.B. 1957. E f f e c t s of 6 d i a z o 5 oxs L - n o r l e u c i n e ' (DON) on t h e r a t l i t t e r i n u t e r o . P r o c . Soc. Exp. B i o l , and Med. 94: 33-35.  136.  T r a v i s , H.F. 1960. N u t r i t i o n a l s t u d i e s on r a n c h - r a i s e d mink. Ph.D. t h e s i s , M i c h i g a n S t a t e U n i v e r s i t y , Ann Arbor Mich.  137.  V e l a r d o , J.T., N.M. Raney, B.G. Smith, and S.H. S t u r g i s . E f f e c t s o f v a r i o u s s t e r o i d s on g e s t a t i o n and l i t t e r s in rats. F e r t i l and S t e r i l . 7: 301-311.  138.  Whitney, R. and H.O. B u r d i c k . 1936. T u b e - l o c k i n g of ova by o e s t r o g e n i c substances. E n d o c r i n o l o g y . 20: 643-647.  139.  Whitney, R. and H.O. B u r d i c k . 1938. A c c e l e r a t i o n o f t h e r a t e of passage of f e r t i l i z e d ova through the F a l l o p i a n tubes of r a b b i t s by massive i n j e c t i o n s o f progynon-B. E n d o c r i n o l o g y 22: 639-642.  1956. size  140.  Whitten, W.K. 1957. The e f f e c t o f p r o g e s t e r o n e on the d e v e l o p ment of mouse eggs i n v i t r o . J . E n d o c r i n . 16: 80-85.  141.  W i n t e n b e r g e r - T o r r e s , S. 1961. Mouvements des trompes e t p r o g r e s s i o n des oeufs chez l a b r e b i s . Ann. B i o l . Anim. B i o c h . Biophys. 1:121-130.  142.  Wright, J.F. and H.R. S e i b o l d . 1958. E s t r o g e n c o n t a m i n a t i o n of p e l l e t e d f e e d f o r l a b o r a t o r y animals - e f f e c t on guinea p i g r e p r o d u c t i o n . J . Amer. Vet. Med. A s s . 132: 258-261.  76.  143.  Yochim, J.M. and V . J . DeFeo. 1962. C o n t r o l of d e c i d u a l growth i n the r a t by s t e r o i d hormones o f the ovary. Endocrinology. 71: 134-142.  144.  Yochim, J.M. and V . J . DeFeo. 1963. Hormonal c o n t r o l of the o n s e t , magnitude and d u r a t i o n of u t e r i n e s e n s i t i v i t y i n the r a t by s t e r o i d hormones of the ovary. Endocrinology. 72: 317-326.  145.  Zarrow, M.X., J.M. Y o s h i n and J . L . McCarthy. 1964. Experimental Endocrinology. Academic P r e s s . New York. pp. 40.  XII  APPENDICES  77. APPENDIX I  ANTIESTROGEN EXTRACTION M a t e r i a l t o be assayed  I  Mix w i t h 0.1 N A u t o c l a v e 30 min. a t 15 l b s . p s i  F i l t e r and c o n c e n t r a t e (heat g e n t l y ) C o o l , A d j u s t pH t o 7.2  I P r e c i p i t a t e s o l u b l e p r o t e i n s w i t h 2 v o l s , of 95% e t h y l a l c o h o l ( l e t stand f o r 24 h r s . )  1 Filter  ^  I  V d i s c a r d ppte.  J"  :  Aqueous e t h y l a l c o h o l  mixture  I Concentrate  Extract  T Ether-soluble  ( i n vacuo) pH 4.5  3x w i t h d i e t h y l ether T  extract  (estrogenic) discard  V — Chloroform-soluble extract (Antiestrogenic)  V Concentrate ( i n vacuo) C h l o r o f o r m E x t r a c t Concentrate (C.E.C.)  Aqueous f r a c t i o V n ( a d j u s t pH t o 415) | V E x t r a c t 3x w i t h c h l o r o f o r m [ V Aqueous f r a c t i o n discard :  78.  APPENDIX I I B l o a s s a y of E s t r o g e n i c and A n t i e s t r o g e n i c Compounds  A.  Ovariectomy  The  animal i s p l a c e d i n a j a r and a n e s t h e s i a i s induced  with ether. it  12 : rat  When.the animal has ceased  t o move or s t r u g g l e ,  i s removed and p l a c e d on an o p e r a t i n g board.  may n o t be c l i p p e d from the s i t e o f o p e r a t i o n .  H a i r may o r Usually  a n e s t h e s i a i s c o n t i n u e d w i t h an e t h e r cone. The  area i s cleaned w i t h a l c o h o l and an i n c i s i o n  from  1.0 t o 1.5 cm. l o n g i s made i n the s k i n midway between the last  r i b and the knee about 1 cm. l a t e r a l t o the s p i n a l A second  i n c i s i o n i s made through  i n t o the p e r i t o n e a l c a v i t y .  the muscle l a y e r and  I f the i n c i s i o n i s made c o r r e c t l y ,  the ovary w i l l be seen immediately mass of f a t .  underneath,  embedded i n a  The f a t i s withdrawn, and the ovary i s s e p a r a t e d  and h e l d w i t h a hemostat.  I t may then be cut away and the  u t e r u s r e t u r n e d t o the p e r i t o n e a l c a v i t y . c l o s e d by means of a s u r g i c a l clamp. s i d e may then be removed through B.  muscles.  The i n c i s i o n i s then  The ovary on the o p p o s i t e  a separate  incision.  Bioassay Replacement therapy w i t h estrogens r e s t o r e s the r e p r o d u c t i v e  t r a c t o f o v a r i e c t o m i z e d animals  t o the p r e c a s t r a t e s t a t e .  The  degree o f growth o f the u t e r u s under such therapy i s p r o p o r t i o n a l to the dose hormone a d m i n i s t e r e d w i t h i n l i m i t s .  Thus, the i n c r e a s e  79.  i n u t e r i n e weight s e r v e s as a convenient b i o a s s a y f o r e s t r o g e n s . S e v e r a l compounds i n h i b i t r e p r o d u c t i v e t r a c t and  the a c t i o n of estrogens on  the  can t h e r e f o r e be t e s t e d f o r t h e i r  a n t i e s t r o g e n i c p r o p e r t i e s w i t h a s i m i l a r b i o a s s a y as t h a t used  f o r the  estrogens.  Immature r a t s , weighing have been o v a r i e c t o m i z e d two  approximately  days p r e v i o u s l y , are  i n t o the r e q u i r e d number of groups. subcutaneously. saline  The  (0.9% N a C l ) .  after injection;  60 gm.  and which distributed  A l l i n j e c t i o n s are made  c o n t r o l group i s i n j e c t e d w i t h The  animals  physiological  are k i l l e d w i t h e t h e r 6 hours  the u t e r i are removed, trimmed of f a t , and  weighed r a p i d l y on a t o r s i o n b a l a n c e to the n e a r e s t 0.1 The u t e r i are expressed  as p e r c e n t body  weight.  mg.  80. APPENDIX I I I .  INITIAL & FINAL BODY WEIGHTS OF STUDY I I , PART A  CONTROL An.. Mo. X33 X44 X183 X296 X112  Int. Final Wt. Wt. Sex (gm) (pm) STANDARDS M  M M M M  1450 ' 1440 1220 1813 1570 1563 1950 1969 1720 1397  WHITE AMD PASTEL X27 X85 X75 X122 X107  M  M M M M  1460 1730 1280 1350 1450  1490 1669 1285 1146. 1332  Int. Final Wt. •Wt. An. No. . Sex (,<?m.) (pm) STANDARDS X154 XI3 X37 X38 X152  M  M M M  M  M M M M M  1230 • 1285 1480 1318 1490 1516 1550 1509 1670 1669  1670 1530 1660 1710 1510  An. No.  1627 . X274 1512 X288 1689 X223 1875 X281 1650 X255  X42 X299 X291 X306 X300  M M M • M M  1740 2030 1460 1280 2030  1812 2025 1687 1213 2009  M M  M M M  1340 1544 1380 1436 1760 1882 1250 ' 1269 1220 1179  1540 1200 2080 1980 1750  M M M M M  1598 1418 2154 2250 1754  An. No. DM33 X164 DM20 W14 W13  X293 X298 X214 1018 W16  M  M M M M  820 1850 1560 1670 1910  M M M ;M M  .M M M M JM  1540 1500 1520 1250 1520  2060 1200 1490. 1380 1720  2155 1182 1544 1395 1821  An. No. X142 X172 X192 X194 XI9 5  1810 2063 1774 1858 2056  DM31 DM29 W20 W19 DM34  M  M M  M M  1650 1780 1620 1700 1820  X187 X186 'D.1 W9 Dtlll  M M M M M.  1840 1920 1710 1280 1320  1740 1470 1380 1420 1720  M M M • M M  175V 1372 1444 1563 1854  VJHITES 1646 X3 2480 X300 1686 X61 1739 - DM15 1753 LI 34  3.990 1810 1350 1490 1890  M.  M M  M M  2044 1751 1470 1495 2060  PASTELS  PASTELS 1538. 1520 1591 1154 1586  -ID  Int. Final Wt. wt-. Sex (gm) (pm) STANDARDS  WHITES .  PASTELS X62 X63 X50 X49 X102  M-  Int. Final Wt. Wt. Sex (pm) (gm) STANDARDS  WHITES  PASTELS X12 Xll X229 X117 X58  M-lC  Int. Final Wt. Wt. Sex (gm) (pm) STANDARDS  STANDARD AND WHITE  PASTELS Xfil X65 X65 . X66 X71  M-lB  M-1A  2038 X101 1762 X59 2006 X83 1412 X130 1386 . X67  M MM M M  •  1820 2081 1130 1137 1280 • 1394 1900 2017 1670 1636  81.  0  APPENDIX III. (CONT.)  An. No.  Sex  Int. Final 'ft. Wt. (gm) (gm)  GREY BLUE g STANDARD X185 X7 X242 X216 X32  M M M M M  1580 1350 1490 1800 1900  M--IB  M-1A  CONTROL  1569 1297 1584 20811991  Int. An. No.  Sex  (gm)  Final  •wt. . An. (gm) No.  GREY BLUE 8 PASTEL X128 X127 XI3 8 X311 X312  M  M  M M  M  1280 1610 1310 1908 1720  1166 1595 1363 2104 1971  Sex  Int. Final Wt. Wt. (gm) (gm)  GREY BLUE 8 PASTEL X217 X144 X105 XI08 X126.  M M M M M  2750 1800 1690 1590 1210  M--ID  M-1C  2013 1857 1838 1667 1425  An. No.  Sex  Int. Final Wt. Wt. (gm) (gm)  GREY BLUE S PASTEL ' DM16 L140 DM13 DM10 DM9  M M M  M M  1210 1750 1840 1360 1570  1398 1938 1920 1604 1725  An. No.  Sex  Int. Final Wt. Vt. (gm) (gm)  GREY BLUE £ PASTEL W2 X248 X52 X113 X100  M M M M M  1300 1570 1720 1540 1470"  .t >i . -\  1268 • J 1535 •S 1717 ',3 1006 • J 1667  'if.  I  ..  DF8 DF109 DF10 DF79 DF12  F F F F F  1010 810 970 860 850  988 943 1059 928 849  F F F F p  980 740 1150 1060 890  DF104 DF81 DF54 DF52 52  F F F • F F  890 900 960 950 780  986 914 996 1125 956  DF11 DF31 DF4 DF74 DF28  1015 653. 1131 1141 921  E84 F E95 F. DF116 F DF44 F DF175 F  700 810 850 770 930  F F F F  940 1040 1070 1060 870  STANDARDS 960 1097 1169 1108 863  DEL 06 DF55 DF80 DF59 DF57  WHITES  WHITES  WHITES DF15 DF138 E80 DF141 E82  STANDARDS  STANDARDS  STANDARDS  796 1051 955 962 1003  DF17 DF48 ' 51 DF115 D.F89  F F F F F  840 950 890 640 890  F F F F F  1040 1150 910 850 670  DF2 49 DF1 DF99 DF30  WHITES 967 944 1073 619 910.  DF42 DF47 DF117 DF21 D174  F F F F F  930 820 870 1030 820  i  STANDARDS 1164 1125 1015 853 611  F F F'  F F  1020 920 720 800 920  -i  1020 945 429 863 1027  E112 E98 E87 E78 E73  F F F F F  720 990 800 1020 910  "I •f  •J -.i  . WHITES . 921 840 967 1264 979  *  704 914. 835 1013 976  p  82.  APPENDIX I I I  CONTROL An. No.  Sex  Final Wt.  .(pin)  (pm)  An. No.  F F F F F  810 770 695 860 840  Int. Final Wt.' Wt. Sex (pjn) (gm)  An. No.  776 825 664 926 892  W106 F W97 F F "91 F "76 W86 . F  920 810 1080 1060 880  1108 880 1159 1218 928  GREY BLUE £ WHITE  GREY BLUE £ MUTE  DF189 DF187 D.F148 DF184 DF185  Int. Final Wt. Wt. Sex (gm) (gm)  F F F F F  920 760 970 870 800  M-ID  M-1C An. No.  Int. Final Wt. Wt. Sex (pm) (gm)  An. No.  982 736 1115 956 944  GREY BLUE £ WHITE  DF166 DF212 DF131 DF231 DF239  F F F F  F  •• -  ...: . j  Int. Final r Wt. . Wt. | Sex (gm) (gm) . I PASTELS  PASTELS  PASTELS  PASTELS  PASTELS' DF236 DF240 DF73 DF128 DF229  M-1B  M-1A  Int. Wt.  (CONT.)  777 860 900 768 1000 . 1151 750 740 1010 930  GREY BLUE £ WHITE  DF132 DF152 DF230 DF151 DF135  F F F F F  794 ji 760 900 979 ;$ 840 : 8i4. 740 680 600 ' 359 :i  GREY BLUE £ WHITE  > .4  DF200 F DF111 F DF96 F DF65 F DF62 F  680 810 730 830 1040  652 780 756 '848 1061  DF225 E96 W15 F103 E104  F F F F F  770 .900 1020 1060 880  707 1017 1246 1006 848  DF87 DF86 DF71 DF119 DF216  F 770 1140 F 760 F 8C0 F. 840 F.  782 1123 640 855 806  DF40 F DF37 ' F DF22 F DF147 F DF223 F  860 940 960 1050 880  892 921 895 1011 1025  DF118 DF97 DF61 50 DF69  F F F F F  920 980 860 780 920  699 996 814 799 781  .$ ' ^  J  A P P E N D I X Initial  and F i n a l  I V  Body Weights  of Study I I , P a r t B  DS-67  Ration  Int. Wt. (gm.)  M-2A Final Wt. (gm.)  Int. Wt. (gm. )  Final Wt. (gm.)  Cage No.  An. No.  762  31  Y627  F  460  678  370  864  32  Y626  F  497  700  F  570  1091  33  Y654  F  544  770  Y185  F  629  1061  34  Y655  F  578  857  5  Y603  F  467  781  35  Y51  F  563  580  6  Y52  F  535  687  36  Y354  F  530  747  7  Y662  F  438  899  37  Y50  F  582  605  8  Y634  F  515  814  38  Y40  •F  531  755  9  Y74  F.  668  776  39  Y39  F  491  800  10  Y628  F  484  870  40  Y3  F  560  746.  11  Y180  F  538  793  41  Y656  F  488  658  Y81  F  525  834  Y657  F  583  992  Y621  F  459  866  Y641  F  427  606  Y622  F  424  686  Y642  F  420  687  Y29  F  548  950  Y637  F  367  596  Y33  F  584  1100  Y638  F  372  550  Y581  F  436  653  Y644  F  466  781  Y582  F  452  687  Y645  F  466  734  Y357  F  460  817  Y630  F  496  978  Y358  F  436  736  Y631  F  456  1035  16  Y186  M  751  1930  46  Y8  M  820  1725  17  Y226  M  625  1529  47  Y629  M  642  1438  Cage NO:. .  An. No.  1  Y227  F  597  2  Y619  F  3  Y225  4,  12  13  14  15  Sex  42  43  44  45  Sex  Int. Wt. (gm.)  Cage No.  An. No.  18  Y184  M  548  19  Y159  M  20  Y127  21  Final Wt. (gm.)  Int. Wt. (gm.)  Final Wt. (gm.)  Cage No.  An. No.  1495  48  Y136  M  674  1652  524  1428  49  Y368  M  561  1445  M  620  1503  50  Y143  M  474  1073  Y623  M  412  1143  51  Y643  M  517  1216  22  Y79  M  668  1630  52  Y644  M  563  1121  23  Y633  M  494  1660  53  Y652  M  600  1091  24  Y170  M  590  1673  54  Y663  M  473  1295  25  Y593  M  604  1558  55  Y653  M  668  996  26  Y583  M  668  1677  56  Y646  M  496  1117  Y584  M  737  1510  Y647  M  540  1293  Y223  M  622  1273  Y18  M  695  1484  Y224  M  594  1306  Y19  M  698  1296  Y179  M  415  1159  Y21  M  645  1357  Y180  M  613  1225  Y22  M  724  1566  Y113  M  378  Y4  M  739  1444  Y114  M  606  1313  Y13  M  660  1247  Y48  M  781  1931  Y31  M  778  1343  Y49  M  758  1942  Y32  M  917  2014  27  28  29  30  Sex  Ration  800  57  58  59  60  M-2B  Sex  M-2C  61  Y353  F  542  832  91  Y373  F  529  1072  62  Y640  F  489  554  92  Y372  F  432  885  63  Y639  F  476  628  93  Y401  F  530  888  64  Y632  F.  497  1035  94  Y411  F  457  886  65  Y602  F  496  845  95  Y481  F  491  786  Int. Wt. (gm. )  Final Wt. (gm.)  Cage No.  An. No.  F  559  945  96  Y517  Y600  F  524  821  97  68  Y586  F  480  832  69  Y47  F  556  70  Y15  F  71  Y598  Int. Wt. (gm.)  Final Wt. (gm.)  F  550  870  Y271  F  456  717  98  Y279  F  485  1030  979  99  Y278  F  515  1052  598  798  100  Y460  F  415  734  F  517  849  101  Y479  F  458  748  Y599  F  494  704  Y480  F  516  856  Y594  F  455  851  Y515  F  482  720  Y595  F  492  650  Y516  F  477  875  Y591  F  535  808  Y269  F  505  954  Y592  F  465  808  Y270  F  414  795  Y45  F  501  769  Y293  F  521  854  Y46  F  627  Y294  F  524  963  Yll  F  574  884  Y431  F  453  657  Y12  F  450  714  Y432  F."  465  746  76  Y363  M  616  1390  106  Y435  M  626  1305  77  Y648  M  512  1222  107  Y487  M  678  1210  78  Y369  M  529  1882  108  Y486  M  618  1225  79  Y590  M  624  1550  109  Y520  M  743  1472  80  Y589  M  618  1502  110  Y312  M  629  1460  81  Y20  M  700  1387  111  Y560  M  740  1460  82  Y10  M  895  1651  112  Y36  M  909  2094  83  Y9  M  736  1336  113  Y44  M  695  1622  84  Y14  M  952  1367  114  Y43  M  633  1190  Cage No.  An. No.  66  Y601  67  72  73  74  75  Sex  102  103  104  1070 105  Sex  Int. Wt. (gm.)  Cage No.  An. No.  85  Y123  M  700  86  Y359  M  Y360 87  88  89  90  Final Wt. (gm.)  Int. Wt. (gm.)  Final Wt. (gm.)  Cage No.  An. No.  Sex  1537  115  Y299  M  616  629  1268  116  Y518  M  658  M  583  1383  Y519  M  745  1440  Y361  M  629  1480  Y297  M  634  1008  Y362  M  619  1156  Y298  M  716  1197  Y658  M  705  1436  Y484  M  602  1022  Y659  M  867  1796  Y485  M  694  1515  Y617  M  478  1293  Y433  M  630  1425  Y618  M  497  1398  Y434  M  656  1415  Y587  M  539  1258  Y558  M  692  1306  Y588  M  566  1330  Y559  M  745  1322  Ration  Sex  117  118  119  120  M-2D  1229 897  M-2E  121  Y422  523  864  151  Y414  F  470  729  122  Y47I  554  765  152  Y415  F  507  719  123  Y548  556  705  153  Y416  F  500  755  124  Y335  535  786  154  Y417  F  455  632  125  Y300  595  848  155  Y418  F  497  635  126  Y379  445  580  156  Y427  F  520  797  127  Y288  511  886  157  Y247  F  362  465  128  Y260  640  817  158  Y428  F  530  774  129  Y253  621  1095  159  Y237  F  604  897  130  Y233  561  745  160  Y511  F  532  856  131  Y350  515  575  161  Y37  F  530  753  Y351  555  470  Y38  F  535  623  Int. Wt. (gm.)  Final Wt. (gm.)  Cage No.  An. No.  F  506  645  162  Y349  F  560  885  Y276  F  547  873  Y277  F  457  758  Y505  F  568  702  Y506  F  624  872  Y279  F  523  997  Y280  F  399  740  136  Y541  M  767  1592  166  Y470  M  676  1103  137  Y352  M  710  1585  167  Y496  M  852  1546  138  Y403  M  940  1606  168  Y576  M  834  1495  139  Y419  M  617  1147  169  Y72  M  720  1155  140  Y340  M  480  897  170  Y238  M  746  1469  141  Y334  M  906  1632  171  Y447  M  666  1510  142  Y550  M  808  1462  172  Y240  M  816  1434  143  Y305  M  640  1212  173  Y579  M  603  1473  144  Y336  M  836  1600  174  Y234  M  888  1560  145  Y553  M  600  1300  175  Y465  M  717  1343  146  Y399  M  743  1198  176  Y41  M  800  1186  Y400  M  840  1294  Y42  M  730  1100  Y507  M  775  1119  Y310  M  550  689  Y508  M  797  1595  Y311  M  590  739  Y301  M  898  1502  Y248  M  626  1374  Y302  M  947  1389  Y254  M  744  1427  Cage No.  An. No.  132  Y348  133  134  135  147  148  Sex  163  164  165  177  178  Int. Wt. (gm.)  Final Wt. (gm.)  Y624  535  844  Y625  490  777  Y235  528  747  Y236  525  802  Y561  625  820  Y562  687  865  Y239  577  735  Y497  435  612  Sex  Int. Wt. (gm. )  Final Wt. (gm.)  Cage No.  An. No.  Sex  149  Y28  M  848  1248  Y30  M  895  1443  Y34  M  854  1625  Y35  M  996  1869  150  Ration  M-2F  181  Y585  F  475  723  182  Y494  F  565  729  183  Y578  F  506  700  184  Y577  F  451  694  185  Y462  F  560  515  186  Y461  F  475  784  187  Y495  F  638  817  188  Y370  F  552  902  189  Y241  F  573  804  190  Y378  F  544  613  191  Y249  F  565  852  Y250  F  564  850  Yl  F  568  620  Y2  F  696  653  Y251  F  490  817  Y252  F  530  872  Y286  F  436  726  Y287  F  527  831  Y303  F  581  882  Y304  F  577  857  192  193  194  195  Int. Wt. (gm.)  Final Wt. (gm.)  Cage No.  An. No.  179  Y429  M  835  1683  Y430  M  674  1129  Y295  M  768  1243  Y296  M  828  1174  180  Sex  Int. Wt. (gm.)  Final Wt. (gm- )  Cage No.  An. No.  196  Y242  M  785  2028  197  Y668  M  470  1535  198  Y261  M  958  1372  199  Y263  M  947  1453  200  Y620  M  568  1028  201  Y463  M  591  1430  202  Y259  M  796  1383  203  Y16  M  908  1712  204  Y262  M  918  1393  205  Y73  M  873  1475  206  Y65  M  833  1537  Y66  M  898  1581  Y285  M  664  1388  Y284  M  822  1396  Y490  M  799  1517  Y491  M  777  1420  Y282  M  714  1297  Y283  M  820  1138  Y492  M  689  1429  Y493  M  720  2026  207  208  209  210  Sex  90.  A P P E N D I X Digestibility  V  Trial  I  Apparent D i g e s t i b i l i t y Dry M a t t e r  Ration  T o t a l Feed Consumption (gms. )  T o t a l Faeces Excreted (gms.)  Males Females  Males Females  Percent D.M. Feed Faeces Males Females  T o t a l D.M. Consumed (gms. )  T o t a l D.M. Excreted (gms. )  Males Females  Males Females  A.D.M. (gms. )  Percent D.M. Digestibility  Males Females  Males Females  M2-A  1134  622  564  203  39.93  35.21 33.71  452.81 248.36  198.58 68.43  254.23 179.93  56.14 72.45  M2-B  956  405  353  103  35.43  30.47 38.44  338.71 143.49  107.56 38.59  231.15 104.90  68.24 73.11  M2-C  681  521  238  166  34.87  33.71 35.63  237.46 181.67  80.23 59.15  157.23 122.52  66.21 67.44  M2-D  673  512  285  127  37.92  28.56 50.72  255.20 194.15  81.40 64.41  173.80 129.74  68.10 66.82  M2-E  521  435  258  122  37.80  27.56 39.38  196.94 164.43  71.10 48.04  125.84 116.39  63.90 70.78  M2-F  872  546  233  184  34.99  39.73 36.26  305.11 191.05  92.57 66.72  212.54 124.33  69.66 65.08  Control  788  440  363  132  37.10  27.32 38.68  292.35 163.24  99.17 51.06  193.18 112.18  66.08 68.20  91.  APPENDIX V (CONTD)  Digestibility  Trial  Apparent D i g e s t i b l e  Ration  M2-A  ,  T o t a l Feed Consumption (gms.)  T o t a l Faeces Excreted  Males Females  Males Females  Percent Feed  Nitrogen Faeces Males  Females  I  Nitrogen  T o t a l Nitrogen Consum ed (gm 3 . )  Total Nitrogen Excreted (gms. )  Males  Males  Females  Females  Nitrogen (gms. ) Males  Percen t N i t r o g en Digest i b i l i t y  Females  Males  Females  1134  622  564  203  6.2317  3.8688  4.1214  70.667  38.761  21.820  8.366  48.847  30.395  69.12  78.42  M2-B  956  405  353  103  7.3620  5.1986  5.8816  70.381  29.816  18.351  6.058  52.030  23.758  73.93  79.68  M2-C  681  521  238  166  8.6889  5.0844  5.3422  59.171  45.269  12.101  8.868  47.070  36.401  79.55  80.41  M2-D  673  512  285  127  7.6954  5.0071  4.6981  51.790  39.400  14.270  5.967  37.520  33.433  72.45  84.86  M2-E  521  435  258  122  7.2009  5.0969  4.7645  37.517  31.324  13.150  5.813  24.367  25.511  64.95  81.44  M2-F  872  546  233  184  7.6599  5.2286  5.3472  66.794  41.823  12.183  9.839  54.611  31.984  81.76  76.47  Control  788  440  363  132  6.7881  4.1253  3.9220  53.490  29.868  14.975  5.177  38.515  24.691  72.00  82.67  92. APPENDIX V (CONTD) Digestibility  Trial  I  Apparent D i g e s t i b l e E t h e r E x t r a c t  Ration  T o t a l Feed Consumption (gm. )  T o t a l Faeces Excreted (gm. )  Males Females  Males Females  Feed  % Ether Extract Faeces Males  Females  T o t a l Fats Consumed (gm. )  T o t a l Fats Excreted (gm. )  Males  Females  Males  Females  Ether Extract (gm. ) Males  Percent E t h e r Extract Digestibility  Females  Males  Females  M2-A  1134  622  564  203  18.2740  8.3723  7.3908  207.23  113.66  47.23  15.00  160.00  98.66  77.21  86.80  M2-.B  956  405  353  103  18.3785  8.6536  4.5343  175.70  74.43  30.55  4.67  145.15  69.76  82.61  93.73  M2-C  681  521  238  166  18.9370  6.3598  4.5188  128.96  98.66  15.14  7.50  113.82  91.16  88.26  92.40  M2-D  673  512  285  127  20.3758  13.2306  4.4409  137.13  104.32  37.71  5.64  99.42  98.68  72.50  94.59  M2-E  521  435  258  122  19.4598  15.0401  5.2756  101.39  84.65  38.80  6.44  62.59  78.21  61.73  92.39  M2-F  872  546  233  184  27.7775  8.3507  7.7840  198.62  124.37  19.46  14.32  179.16 110.05  90.20  88.49  Control  788  440  363  132  20.5512  12.8800  6.7892  161.94  90.43  46.75  8.96  81.47  71.13  90.09  115.19  93. APPENDIX V (CONTD)  Digestibility  Trial  I  Apparent D i g e s t i b l e Energy  T o t a l Feed Consumption (gm. )  T o t a l Faeces Excreted  Males  Females  Males Females  M2-A  1134  622  564  203  5.2634  M2-B  956  405  353  103  M2-C  681  521  238  M2-D  673  512  M2-E  521  M2-F  Control  Ra t i on  C a l o r i c d e n s i t y (Cal .)Tbtal Energy Consumed (KCal.) Feed Faeces Males  Females  Males  Females  T o t a l Energy Excreted (KCal.) Males  Energy (KCal.) Females  Males  Females  3.6857 3". 5887." 5969.04 3274.02 2078.73  728.51 3890.31 2545.51  65.17  77.75  5.0656  3.5722 3.4640  4842.71 2051.57 1260.99  356.79 3581.72 1694.78  73.96  82.61  166  5.0281  3.6163 3.3118  3424.14 2619.64  860.68  549.76 2563.46 2069.88  74.86  79.01  285  127  5.0741  3.1622 3.3780  3414.87 2597.94  901.23  429.01 2513.64 2168.93  73.61  83.49  435  258  122  5.0614  3.5702 3.3068  2636.99 2201.71  921.11  403.43 1715.88 1798.28  65.07  81.68  872  546  233  184  5.0465  3.2954 3.2722  4400.55 2755.39  767.83  602.08 3632.72 2153.31  82.55  78.15  788  440  363  132  5.1772  3.6167 3.2778  4079.63 2277.97 1312.86  432.67 2766.77 1845.30  67.82  81.01  Females  Males  Percent D i g . Energy  APPENDIX V (CONTD)  Digestibility Nitrogen  Ration  Trial  I  Retention  Total Nitrogen Consumed (gm. )  Total Faecal Nitrogen (gm. )  Average Concentration U r i n a r y N. (mgN/ml.)  Total Urine Excreted (ml.)  Total Urinary Nitrogen (gm. )  Total Nitrogen Excreted (gm. )  Nitrogen Balance (gm. )  Males Females  Males Females  Males Females  Males Females  Males Females  Males Females  Males Females  M-2A  70.67  38.76  21.82  8.37  43.52  34.37  389.5  198.0  16.95  6.81  38.77  15.18  +31.90 +23.58  M-2B  70.38  29.82  18.35  6.06  40.19  34.77  385.5  152.0  15.49  5.29  33.84  11.35  +36.54 +18.47  M-2C  59.17  45.27  12.10  8.87  29.74  34.33  394.5  218.0  11.73  7.48  23.83  16.35  +35.34 +28.92  M-2D  51.79  39.40  14.27  5.97  41.96  37.20  255.5  194.0  10.72  7.22  24.99  13.19  +26.80 +26.21  M-2E  37.52  31.32  13.15  5.81  41.00  32.99  217.0  164.0  .9.90  5.41  23.05  11.22  +14.47 +16.85  M-2F  66.79  41.82  12.18  9.84  39.92  30.64  300.5  209.0  12.00  6.40  24.18  16.24  +42.61  Control  53.49  29.87  14.98  5.18  31.85  27.73  339.5  187.5  10.81  5.20  25.79  10.38  +27.70 +19.49  25.58  95.  APPENDIX VI Digestibility Trial II  Ration  T o t a l Peed Consumption (gms.) Males  T o t a l Faeces Excreted (gms.)  Females Males  Females  Apparent D i g e s t i b l e  Dry Matter  Per cent Feed  T o t a l D.M. Consumed (gms.)  %  D.M. Faeces  Ma^es Females  T o t a l D.M. Excreted (gms.)  Males  Females Males  A.D.M. (gms.)  Females Males Females Males) Females  M2A  300  570  133  270  39.86 30.32 33.32  90.96  227.20  40.33  89.96  M2B  472  291  146  98  38.19 34.48 30.66  180. 26  111.13  50.34  30.05 L29.92  M2C  483  331  159  131  41.27 34.50 32.68  199.33  136.60  54.86  42.81 144.47  M2D  676  603  203  213  38.09 34.68 30.57  257.49  229.68  M2E  482  561  143  183  38.00 32.53 37.49  183.16  M2F  605  206  285  85  38.11 34.34 38.26  230.57  592  348  171  36.79 30.64 35.78  387.03  C o n t r o l 1052  Percent D.M. Digestibility  55.66  60.40  81.08  72.07  72.96  93.'79  72.48  68.66  70.40  65.11 187.09 164.57  72.66  71.65  213.18  46.52  68.61 136.64 144.57  74.60  67.82  87.51  97.87  32.52 132.70  45.99  57.55  58.58  61.18 280.40 156.62  72.45  71.91  217.80 106.63  50.63 137.24  96 APPENDIX VI (Contd.) Digestibility Trial Apparent D i g e s t i b l e  Ration  T o t a l Feed Consumption (gms.) Males  l  T o t a l Faeces Excreted (gms.) •  Females  Males  Females  Nitrogen.  j Percent Feed  Nitrogen Faeces  II  (gm) .  Perceio.t : N i t r o gen Diges t i b i l i t y  Females  Males Females  Males Females  Total Nitrogen Total Nitrogen E x c r e t ed Consumed (gm) (gm)  Males Females Males  Females Males  Nitrogen !  M2A  300  570  133  270  5.5201 4.0400 4.0420 16.56  31.47  5.37  10.91  11.19 20.56  67.55 .65.32  M2B  472  291  146  98  6.9138 4.6189 5.1780 32.63  20, 12  6.74  5.07  25.89 15.05  79.33  74. 78  M2C  483  331  159 .  131  7.0612 4.7581 5.4641 34.11  23.37  7.57  7.16  26.54 16.22  77.89  69.37  M2D  676  603  203  213 . 6.2212 4.2046 3.4085 42.06  25.35  8.54  7.26  33.52 18.09  79.71  71.37  M2E  482  561  143  183' 6.9545 4.3091 4.9898 33. 52  24.17  6.16  . 9.13  27.36 15.04  81.62  62.23  605  206  285  85  7.0044 6.3902 4.4968 42.38  13.16  18.21  3.82  24.17  1052  592  348  171  6.3220 4.0911 4.6084 66.51  37.43  14.24  7.88  52.27 29.55  M2F  Control  9.34- ;". 57.02  78.59  70.97-  78.95  97.  APPENDIX VI (Contd) Digestibility Trial II Apparent D i g e s t i b l e E t h e r E x t r a c t  Ration  T o t a l Feed Consunl p t i o n (gms) Males  T o t a l Faeces Excreted (gms)  Females Males  Females  Percent E t h e r Extract Feed Faeces  Diges t . E.E.  T o t a l Fats Excreted (gms)  Ether Extract (gms)  Males Females  Males Females Males Females  L57.43  10.96  26.74  71.90 130.69  24.6411 5.1281 10.1018 116.31 71.71  7.49  9090  108.82  %  Males  T o t a l Fats Consumed (gms)  females Males Females  86.77  83.01  61.81  93.56  86.19  71.17  94.04, 87.67  M2-A  300  570  133  270  M2-B  472  291  146  98  M2-C  483  331  159  131  24.5268 4.4388 7.6408  118.46 81.18  7.06  10.01  111.40  M2-D  676  603  203  213  24.7850 6.8621 9.7553  167.55 149.45 13.93  20.78  153.62 128.67  91.69  86.10  M2-E  482  561  143  183  24.1943 3.4682 6.5267  116.62 135.73  4.96  11.94  111.66 123.79  95.75  91.20  605  206  285  85  24.6602 5.2335 5.5846  149.19  50.80 14.92  4.75  134.27  46.05  90.00  90.65  1052  592  348  171  24.4532 7.691S 5.5401  -. J 257.25 144.76 26.77  '0.47  230.48 135.29  89.59  93.46  M2-F  Control  27.6196 8.2374 9.9047  82.86  %  Appendix VI (Contd)  -  Digestibility T r i a l II  Apparent Digestible Energy Total Feed Total Faeces Consumption Excreted Ration (gm) (gm) Males Fem-?.Males ales  Females  Caloric Density Kcal / gm Feed Faeces Males  Females  Total Energy Consumed (Kcals)  Total Energy Excreted (Kcal)  Males  Females  Males  Females  Males  Females  Males  Females  Digesti ble Energy  A Energy (Kcals)  M2-A  360  570  133  270  5.2223 3.6339 3.7264  1566.69  2976.71  483.31  1006.13  1083.38  1970.58  69.15  66.20  M2-B  472  291  146  98  5.2538 3.6151 3.6993  2479.79  1528.86  527.80  362.53  1951.99  1166.33  78.72  76.29  M2-C  483  331  159  131  5.5210 3.6719 3.5902  2666.64  1827.46  583.83  470.32  2082.81  1357.13  78.11  74.26  M2-D  676  603  203  213  5.2604 3.6952 3.6499  3556.03  3172.02  750.13  777.43  2805.90  2394.59  78.91  75.49  M2-E  482  561  143  183  5.2709 3.6122 3.5957  2540.57  2956.97  516.54  658.01  2024.03  2298.96  79.67  77.75  M2-F  605  206  285  85  5.1897 3.6725 3.4270  3139.77  1069.08  1046.66  291.30  2093.11  777.78  66.66  72.75  Control 1052  592  348  171  5.3590 3.6500 3.4281  5637.67  3172.53  1270.20  586.21  4367.47  2586.32  77.47  81.52  APPENDIX VI (Contd) Digestibility Trial II  Total N Consun:ed  Ration  (gm) 1  Male  Female  -  Nitrogen Retention.  Total Fecal Nitrogen (gm)  Average Concentration Urinary N (mgN/ml)  Male  Female  Male  Female  Male  Female  Male  T o t a l Urine Excreted (ml)  ' Total Urinary N i t r o g ;en (gm)  Total N Excreted (gm)  Nitrogen Balance (gm)  Female  Male  Female  Male  Female  M-2A  16.56  31.47  5.37  10.91  31.40  44.52  165.5  180.0  5.20  8.01  10.57  18.92  +5.99  +12.55  M-2B  32.63  20.12  6.74  5.07  46.13  40.79  169.5  129.5  7.82  5.28  14.56  10.35 +18.07  + 9.77  M-2C  34.11  23.37  7.57  7.16  42.81  39.80  253.5  171.5  10.85  6.83  18.42  13.99 +15.69  + 7.68  M-2D  42.06  25.35  8.54  7.26  46.22  38.56  295.0  266.0  13.63  10.26  22.17  17.52 +19.89  + 7.83  M-2E  33.52  24.17  6.16  9.13  35.36  41.99  235.0  176.5 .  8.33  7.41  14.49  16.54 +19.03  + 7.73  M-2F  42.38  13.16  18.21  3.82  37.53  39.80  229.5  126.5  8.61  5.03  26.82  8.85 +15.56  + 4.31  Control  66.51  37.43  14.24  7.88  38.36  37.21  455.5  241.0  17.47  8.97  31.71  16.85 +34.80  +20.58  APPENDIX V I I A n a l y s i s o f V a r i a n c e Tables, Study I  Bioassay 1  :' E s t r a d i o l and C.E.C.  Source  DF  Mean Sq.  Sum- Sq.  Trts.  2  Error  12  . 3.0702 x 10" -4 2.8420 x 10  Total  14  5.9122 x 10"  B i o a s s a y 2 '.. : Source  Estradiol  DF  1.5351  F x  2.3683 x 10  10 -5 •  6.48  and C.E.C. Mean Sq.  Sum Sq.  Trts/  2  '6.1120 x 10"  Error  15  4.2492 x 10'  Total  17  1.0361 x 10"  -4 3.0560 x 10 -5 2.8328 x 10  F 10.79  B i o a s s a y o f DES. Source Trt.  DF 3  Sum Sq.  Mean Sq.  F  1.054 x.10  3.513 x 10  24.83  Error  20  2.820 x 10"  Total  23  1.337 x 10"'  2  1.415 x 10"  101.  STUDY I I  PART A. 1.  I n i t i a l Body Weight  Source  DF  Sum Sq.  Mean Sq.  Ration  4  111120  27780  Sex  1  24691000  24691000  R-S  4  454920  11373 45191  Error  190  8586200  Total  199  33434000  2.  F 0.61  Gain  Source  DF  Sum Sq.  Mean Sq.  F  Ration  4  210710  52678  2.26  Sex  1  43395  43395  R-S  4  97700  24425 23284  Error  190  4424000  Total  199  4775800  3.  F i n a l Body Weight  Source  DF  Ration  Sum Sq.  Mean Sq.  F  4  619050  154760  2.85  Sex  1  27096000  27096000  R-S  4  274480  68619 54384  Error  190  10333000  Total  199  58322000  PART B. 1.  I n i t i a l Body Weight  Source  DF  Sum Sq.  Mean Sq.  Ration Sex RxS Cage CxR CxS RxSxC Error Total  6 1 6 1 6 1 6 252 279  493510 2326300 202730 167000 45976 16386 163110 2053300 5301500  82252 2326300 33789 167.00 7662.7 16386 27186 8147.9  Source  DE  Sum Sq.  Mean Sq.  Ration Sex RxS Cage CxR CxS RxCxS Error Total  6 1 6 1 6 1 6 252 279  1263900 12755000 530100 129950 301750 163980 51761 6771000 21968000  210650 12755000 88350 129950 50292 163980 8626.9 26869  2.  3.  F 10.09 285.51 4.15 0.02 0.94 2.01 3.34  Gain F 7.84 474.72 3.29 4.84 1.87 6.10 0.32  F i n a l Body Weight  Source  DF  Sum Sq.  Mean Sq.  Ration Sex RxS Cage CxR CxS RxCxS Error Total  6 1 6 1 6 1 6 252 279  549860 25990000 320740 121760 326840 75928 208880 9477400 37072000  91644 25990000 53457 121760 5447.3 75928 34813 37609  F  .  244 691.07 1.42 3.24 1.45 2.02 0.93  103.  A n a l y s i s of Covariance.  Source  d.f.  Trts.  39  Error Total  Study I I .  P a r t B.  SSx  SP  SSy  SS'y  MSfy  F  845520  495465  42513335  -29557767  -760455  12.08  239  42076681  49022592  42071512  -15043604  -62944  278  42922201  49518057  42513335  -14614163  

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